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遗传学(第3版) 刘祖洞、乔守怡、吴燕华、赵寿元高等教育出版社 (2013-01)课后习题答案10

Chapter 10 Messenger RNA Cap Methylation in Vesicular Stomatitis Virus, a Prototype of Non‐Segmented Negative‐Sense RNA Virus

Jianrong Li and Yu Zhang

Additional information is available at the end of the chapter

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1. Introduction

The non‐segmented negative‐sense (NNS) RNA viruses encompass a wide range of signifi‐cant human, animal, and plant pathogens including several National Institute of Allergy and Infectious Diseases (NIAID) Category A and C biodefense pathogens. The NNS RNA viruses are classified into four families: the Rhabdoviridae, as exemplified by rabies virus and vesicular stomatitis virus (VSV); the Paramyxoviridae, as exemplified by human respiratory syncytial virus (RSV), human metapneumovirus (hMPV), human parainfluenza virus type 3 (PIV3), measles virus, mump virus, Newcastle disease virus (NDV), Nipah virus, and Hen‐dra virus; the Filoviridae, as exemplified by Ebola and Marburg viruses; and the Bornaviridae, as exemplified by Borna disease virus. For many of these viruses, there are no effective vac‐cines or anti-viral drugs. RSV, hMPV, and PIV3 account for more than 70% of acute viral respiratory diseases, especially in infants, children, and the elderly [1, 2]. hPIV 1-3 have been recognized as the causative agents of croup since the late 1950’s [3]. In addition, measles re‐mains a major killer of children worldwide, despite successful vaccination programs in de‐veloped countries [4]. The most virulent strains of NDV, the viscerotropic velogenic strains (often called “exotic” NDV), are classified as High Consequence Livestock Pathogens by US‐DA due to their potential as agents of agricultural bioterrorism [5].

Messenger RNA modification is the essential issue in NNS RNA virus gene expression and replication. During viral RNA synthesis, NNS RNA viruses produce capped, methylated, and polyadenylated mRNAs [6-8]. Cap formation is essential for mRNA stability, efficient transla‐tion, and gene expression [9-11]. It is now firmly established that mRNA capping and methyla‐tion in NNS RNA viruses evolves in a mechanism distinct to their hosts [12-19]. Thus, mRNA cap formation is an attractive antiviral target for NNS RNA viruses. For decades, VSV has been

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used as a model to understand the replication and gene expression of NNS RNA viruses. Most of our understanding of mRNA modifications of NNS RNA viruses comes from studies of VSV, a prototype of the Rhabdoviridae family. Using VSV as a model, we will discuss (i) the un‐usual mechanism of mRNA capping and cap methylation; (ii) the impact of viral mRNA cap methylation in viral life cycle and viral pathogenesis; and (iii) the applications of viral mRNA cap methylation in the development of novel vaccines and broadly-active anti-viral agents.

2. Overview diagram of VSV mRNA synthesis and modifications

2.1. The structure of VSV virions

VSV virions are bullet-shaped particles 170 nm in length and 80 nm in diameter (Fig.1). Among NNS RNA viruses, VSV has the simplest RNA genome consisting of 11,161 nucleotides (nt) or‐ganized into five VSV genes encoding nucleocapsid (N), phospho- (P), matrix (M), glyco- (G),and large (L) proteins, and leader and trailer regulatory sequences arranged in the order 3’-(leader), N, P, M, G, L, (trailer)-5’[20-23]. Like all NNS RNA viruses, the genome is encapsidated with the N protein to form a nuclease-resistant helical N-RNA complex that is the functional template for mRNA synthesis as well as genomic RNA replication. The N-RNA complex is tight‐ly associated with the viral RNA-dependent RNA polymerase (RdRp), which is comprised of the 241-kDa L protein catalytic subunit and the 29-kDa essential P protein cofactor, and results in the assembly of a viral ribonucleoprotein (RNP) complex [24, 25]. This structure contains the minimum virus encoded components of the VSV RNA synthesis machinery [26]. The RNP com‐plex is further surrounded by the M protein which plays a crucial role in virus assembly, bud‐ding, and maintenance of the structural integrity of the virus particle [27]. The outer membrane of virion is the envelope composed of a cellular lipid bilayer. The transmembrane G protein is anchored in the viral envelope, which is essential for receptor binding and cell entry [28].

2.2. VSV life cycle

The overview picture of VSV life cycle is depicted in Fig.2. Upon attaching to an unknown cell receptor(s), VSV enters host cells via receptor mediated endocytosis [29]. Following low pH triggered fusion and uncoating, the RNP complex is delivered into the cytoplasm where RNA synthesis and viral replication occur [30]. During primary transcription, the input RdRp recog‐nizes the specific signals in the N-RNA template to transcribe six discrete RNAs: a 47-nucleo‐tide leader RNA (Le+), which is neither capped nor polyadenylated, and 5 mRNAs that are capped and methylated at the 5’ end and polyadenylated at the 3’end. These mature mRNAs are then translated by host ribosomes to yield functional viral proteins which are required for viral genome replication. During replication, the RdRP initiates at the extreme 3’ end of the ge‐nome and synthesizes a full-length complementary antigenome, which subsequently serves as template for synthesis of full-length progeny genomes. These progeny genomes can then be utilized as templates for secondary transcription, or assembled into infectious particles. Final‐ly, viral proteins and genomic RNA are assembled into complete virus particles and the virus exits the cell by budding through the plasma membrane.

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Figure 1. VSV virion structure and genome organization. VSV encodes five structural proteins : nucleocapsid (N),phospho- (P), matrix (M), glyco- (G), and large (L) proteins. The VSV genome is arranged in the order 3’-(leader), N, P,

M, G, L, (trailer)-5’.

Figure 2. Overview diagram of VSV life cycle . Steps of virus life cycle: attachment, endocytosis, uncoating, genome replication, mRNA transcription, viral protein translation, viral assembly, and budding are shown.

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2.3. VSV mRNA synthesis and modifications

Our current understanding of VSV mRNA synthesis and modification can be summarized as follows. In response to a specific promoter element provided by the genomic leader re‐gion, the polymerase initiates mRNA synthesis at the first gene-start sequence to synthesize N gene. The nascent mRNA is capped through an unconventional mechanism in which the GDP: polyribonucleotidyltransferase (PRNTase) of L transfers a monophosphate RNA onto a GDP acceptor through a covalent protein-RNA intermediate [12, 13, 16]. Following cap ad‐dition, VSV mRNAs are sequentially methylated at ribose 2’-O position and G-N-7 position,which is distinct from all known methylation reactions [17, 18]. Unlike traditional cap form‐ing enzymes, the VSV capping and methylation machinery requires cis-acting signals in the RNA [12, 18, 31, 32]. When encountering a gene-end sequence, L polyadenylates and termi‐nates mRNA synthesis by a programmed stuttering of the polymerase on a U7 tract [33, 34].Termination at the end of the N gene is essential for the polymerase to initiate synthesis at the start of the next gene, to produce the P mRNA. During transcription, the RdRp complex transcribes the viral genome into five mRNAs in a sequential and gradient manner, such that 3′ proximal genes are transcribed more abundantly than 3′ distal genes [21-23]. This gradient of transcription reflects a poorly understood transcriptional attenuation event that is localized to the gene junction regions. Using this fashion, VSV produces five capped, me‐thylated, and polyadenylated mRNAs, N, P, M, G, and L.

3. Large (L) polymerase protein, the multifunctional protein that

modifies viral mRNA

All NNS RNA viruses encode a large (L) polymerase protein, a multifunctional protein rang‐ing from 220-250kDa in molecular weight. The L protein contains enzymatic activities for nu‐cleotide polymerization, mRNA cap addition, cap methylation, and polyadenylation. To date,the structure of L protein, or L protein fragments, has not been determined for any of the NNS RNA viruses. Amino acid sequence alignment between the L proteins of representative mem‐bers of each family within NNS RNA viruses has identified six conserved regions numbered I to VI (CRs I–VI) (Fig.3) [35]. Thus, there is a general assent that the enzymatic activities of L pro‐tein are located in these conserved regions. For the last four decades, VSV L protein has been used a model to understand the different activities of NNS RNA virus L proteins because it is the only member of this order of viruses for which robust transcription can be reconstituted in vitro [6, 7, 21, 36]. In addition, the VSV L protein can be highly expressed in recombinant expres‐sion systems, such as E.coli and insect cells. The purified VSV L protein retains all the enzymat‐ic activities that can modify short virus-specific mRNA in trans [12, 16, 18, 37]. In recent years,many breakthroughs have been made in the characterization of the function of VSV L protein and the enzymatic activities have been mapped at the single amino acid level (Fig.3). Within the primary sequence of L are six conserved regions shared among all NNS RNA virus L proteins.The RdRP activity has been identified in CR III and this region is also required for polyadenyla‐tion [38-40]. Consistent with this, a GDN motif is conserved in CR III of all NNS RNA virus L proteins and is functionally equivalent to the GDD polymerization motif characteristic of posi‐Methylation - From DNA, RNA and Histones to Diseases and Treatment

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tive strand RdRPs. Mutations to the GDN motif of the VSV L protein inactivated polymerase function [40]. The mRNA capping enzyme maps to CR V [13, 16], and the capping activities of L differ from those of other viruses and their eukaryotic hosts. Specifically, an RNA:GDP PRNTase activity present within CR V transfers 5′ monophosphate RNA onto a GDP acceptor through a covalent L-pRNA intermediate [12, 13, 16]. The mRNA cap methyltransferases (MTase) map to CR VI [14, 17, 19]. Like the unconventional capping enzyme, methylation of the VSV mRNA cap structure is also unique in that mRNA cap is modified by a dual specificity MTase activity within CR VI whereby ribose 2′-O methylation precedes and facilitates subse‐quent guanine-N-7 (G-N-7) methylation [17, 18]. Although functions have not yet to be as‐signed to the other three conserved regions (CRs I, II, and IV), experiments with Sendai virus

(SeV) have implicated CR I in binding P protein and CR II in binding the RNA template [41, 42].Figure 3. Conserved regions in L proteins of NNS RNA viruses . Six conserved regions numbered I to VI (CRs I–VI) in L pro‐tein are shown. Signature motifs for nucleotide polymerization, mRNA cap addition, and cap methylation are shown.

The location of the nucleotide polymerization, capping, and cap methylation activities with‐in separate regions of L has led to the notion that L protein may be organized as a series of independent structural domains. Consistent with this idea, a fragment containing CRs V and VI of the SeV L protein were expressed independently and shown to retain the ability to methylate short RNAs that corresponded to the 5′ end of SeV mRNA [43]. In addition, re‐combinant VSV and measles virus can be recovered from infectious cDNA clones by insert‐ing the coding sequence of green fluorescent protein between CR V and VI in L gene,suggesting that L protein folds and functions as a series of independent globular domains

[44, 45]. Interestingly, mutations to a variable region between CRs V and VI (residues 1450–1481) affect mRNA cap MTase activity, feasibly suggesting that mutation to this hinge re‐gion may affect a conformational change in CR VI [46]. More recently, the molecular archi‐tecture of VSV L protein has been revealed using negative stain electron microscopy (EM) in combination with proteolytic digestion and deletion mapping [37]. It was found that VSV L protein is organized into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Interestingly, upon binding to P protein, L protein undergoes a significant structural rearrangement that may facilitate the coordination between mRNA synthesis and capping apparatus [37, 47].

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4. An unconventional mRNA capping mechanism in VSV

4.1. Conventional mechanism of mRNA capping in eukaryotic cells

In eukaryotic cells, capping of mRNA is an early posttranscriptional event that is essential for subsequent processing, nuclear export, stability, and translation of mRNA [11, 48]. Cap formation is mediated by a series of enzymatic reactions (Fig.4A). First, the 5’ triphosphate end of the nascent mRNA chain (5’pppN-RNA) is hydrolyzed by an RNA triphosphatase (RTPase) to yield the diphosphate 5’ ppN-RNA. Second, an RNA guanylyltransferase (GTase) reacts with GTP to form a covalent enzyme-GMP intermediate and transfers GMP to 5’ppN-RNA via a 5′-5′ triphosphate linkage to yield 5’ GpppN-RNA. Typically, RNA GTases contain a signature Kx[D/N]G motif that functions as an active site for the capping reaction [11, 48, 49]. A lysine residue within Kx[D/N]G motif forms the enzyme-GMP cova‐lent intermediate, prior to its transfer onto the diphosphate RNA acceptor [11, 48, 50]. This mRNA capping reaction is conserved among all eukaryotes.

Viruses are highly diverse in capping their mRNA. Many DNA viruses (such as vaccinia vi‐rus and baculovirus), double stranded RNA viruses (such as reovirus, rotavirus, and blue‐tongue virus), and single strand positive RNA viruses (such as West Nile virus, Fig.4B)utilize the conventional eukaryotic capping pathway [51-56]. It has been suggested that Kx[V/L/I]S motif serve as the GTase active site for reovirus and rotavirus [57]. Other viruses have evolved different mechanisms for acquiring their cap. For example, influenza virus and hantavirus furnish their mRNA with this structure by a cap-snatching mechanism, in which the viral polymerase steals host cell mRNA caps to prime viral mRNA synthesis [58,59]. The alphaviruses, such as Sindbis, have evolved S-adenosyl-L-methionine(SAM )-de‐pendent GTase activities that utilize distinct motifs (such as HxH motif) to transfer 7m Gp through a covalent histidine Gp intermediate to form the 7m GpppN cap [60].

4.2. An unconventional mRNA capping mechanism in VSV

In the early 1970’s, it was suggested that the cap structure of NNS viral mRNAs was formed by a mechanism which was unique from eukaryotic cap formation. For VSV [6], RSV [61], and spring viremia of carp virus [62], the two italicized phosphates of the 5′G pp p5′NpNpN triphosphate bridge have been shown to be derived from a GDP donor, rather than GMP. However, further studies on this mechanism have been seriously hampered due to the fact that the VSV capping events are tightly coupled to transcription and the capping machinery does not respond to exog‐enous transcripts. In 2007, this unique capping mechanism was revealed using a novel trans cap‐ping assay, in which a short mRNA corresponding to the first 5-nt of VSV gene start sequence was capped by a highly purified L protein in trans [12]. Specifically, capping of VSV mRNA was achieved by a novel polyribonucelotidyltransferase (PRNTase) which transferred a monophos‐phate RNA onto a GDP acceptor through a covalent L-RNA intermediate (Fig. 4C). In the first step, a GTPase associated with the VSV L protein removes the γ-phosphate group of GTP to gen‐erate GDP, an RNA acceptor. In the second step, the PRNTase activity of the L protein specifical‐ly transfers a 5′-monophosphorylated (p-) RNA moiety of pppRNA with the conserved VSV mRNA-start sequence (AACAG) to GDP to yield a GpppA capped mRNA. Interestingly, this Methylation - From DNA, RNA and Histones to Diseases and Treatment

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unusual VSV capping enzyme caps RNA in a sequence specific manner [12]. Specifically, VSV L protein efficiently capped pppApApCpApG (the mRNA gene start sequence), but not pppApCpGpApA (the leader RNA start sequence). In addition, the VSV L protein was not able to cap ppApApCpApG, suggesting that the L protein specifically recognizes the 5′-triphos‐phorylated AACAG. Further mutagenesis analysis has shown that the APuCNG (Pu, purine)sequence acts as a cis

-acting element for the RNA capping reaction for the VSV L protein [12].

Figure 4. Comparison of mRNA cap formation in eukaryotic cells, WNV, and VSV. (A) Cellular mRNA cap forma‐tion. First, pppNNNN-RNA is hydrolyzed by an RNA triphosphatase (RTPase) to yield the diphosphate ppNNNN-RNA.Second, an RNA guanylyltransferase (GTase) transfers GMP to ppNNNN-RNA to yield GpppNNNN-RNA. Third,GpppNNNN-RNA is methylated by G-N-7 MTase to yield 7m GpppNNNN-RNA. Fourth, 7m GpppN-RNA is further methy‐lated by a 2’-O MTase to yield 7m GpppN m NNN-RNA. (B) WNV mRNA cap formation. First, pppNNNN-RNA is hydro‐lyzed by viral NS3 protein (RTPase) to yield ppNNNN-RNA. Second, viral NS5 protein (GTase) transfers GMP to ppNNNN-RNA to yield GpppNNNN-RNA. Third, GpppNNNN-RNA is methylated by N-terminus of NS5 (G-N-7 MTase) to yield 7m GpppNNNN-RNA. Fourth, 7m GpppN-RNA is further methylated by N-terminus of NS5 (2’-O MTase) to yield Messenger RNA Cap Methylation in Vesicular Stomatitis Virus, a Prototype of Non‐Segmented Negative‐Sense RNA

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7m GpppN m NNN-RNA. (C) VSV mRNA cap formation. First, a GTPase (CR V of L protein) removes the γ-phosphate group of GTP to generate GDP. Second, a polyribonucelotidyltransferase (PRNTase) (CR V of L protein) transfers a monophosphate RNA onto a GDP acceptor through a covalent L-RNA intermediate to GpppAACAG-RNA. Third, the cap structure is methylated a 2’-O MTase (CR VI of L protein) to yield GpppA m ACAG-RNA. Fourth, GpppA m ACAG-RNA is further methylated a G-N-7 MTase (CR VI of L protein) to yield 7m GpppA m ACAG-RNA.

It has been a challenge to locate the active site for the novel PRNTase in the 241 kDa L protein.The only suggestive information regarding the location of the capping enzyme in L protein has come from the study of a novel inhibitor of the RSV polymerase which resulted in the synthesis of short uncapped viral RNAs in vitro [63]. Viral mutants resistant to this inhibitor were select‐ed, and the resistance mutations were mapped to CR V, suggesting that CR V of L plays a role in mRNA cap formation. Sequence alignments of this region of L protein identified a total of 17residues that were conserved among the NNS RNA viruses [16]. Guided by this information,an extensive mutagenesis analysis was performed within this region which led to the discov‐ery of a new motif GxxT[n]HR composed of four amino acid residues (G1154A, T1157A,H1227A, and R1228A) in VSV L protein which are essential for mRNA cap formation [16]. In vi‐tro RNA reconstitution assays have shown that these cap defective polymerases synthesized uncapped mRNAs that terminated prematurely. The size of these abortive transcripts ranged from 100 nt up to the full-length N mRNA, although the majority were less than 400 nt. Consis‐tent with their inability to generate capped RNA during in vitro transcription reactions,G1154A, T1157A, H1227A, and R1228A were defective in trans capping of the 5-nt VSV gene start sequence, demonstrating that these amino acids in CR V of L protein are required for mRNA cap addition [16]. Importantly, GxxT[n]HR is highly conserved in the CR V of L pro‐teins of all NNS RNA viruses, including Borna disease virus which replicates in nucleus. Fur‐ther biochemical and mass spectrometric analyses found that H1227 in the conserved GxxT[n]HR motif of the VSV L protein is covalently linked to the 5′-monophosphate end of the RNA through a phosphoamide bond [13]. Therefore, amino acid residue H1227 is the active site of the PRNTase activity. Mutagenesis analysis also found that R1228A and R1228K muta‐tions significantly decreased L-pRNA complex formation activities, suggesting that mutation in R1228 may affect the H1227-RNA intermediate formation [13]. Interestingly, this PRNTase activity was also found in L protein of Chandipura virus (CHPV), a rhabdovirus that is closely related to VSV [64]. Furthermore, mutations to HR motif in L protein of CHPV significantly re‐duced the formation of the L-pRNA covalent intermediates in the PRNTase reaction. These re‐sults demonstrate that this unconventional capping mechanism is conserved in the Rhabdoviridae family. Given the fact the HR motif is highly conserved in L proteins of NNS RNA viruses, it is likely that this novel capping mechanism is not only unique to rhadoviruses, but also may be utilized by other NNS RNA viruses.

5. An unusual mechanism of mRNA cap methylation in VSV

5.1. Conventional mRNA cap methylation in eukaryotic cells

In eukaryotic cells, the capped mRNA (GpppN-RNA) is typically methylated by two steps (Fig.4A) [65-68]. First, the capping guanylate is methylated by a G-N-7 methyltransferase Methylation - From DNA, RNA and Histones to Diseases and Treatment

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(MTase) to yield 7m GpppN-RNA (cap 0). Second, the G-N-7 methylated cap structure can then be further methylated by a ribose-2’-O (2’-O) MTase to yield 7m GpppN m -RNA (cap 1).During mRNA cap methylation, S-adenosyl-L-methionine (SAM) serves as the methyl do‐nor, and the by-product S-adenosyl-homocysteine (SAH) is the competitive inhibitor of the SAM-dependent MTase. These mRNA cap methylation reactions are conserved among all eukaryotes. In this conventional methylation reaction, G-N-7 methylation occurs prior to 2’-O methylation and the two methylase activities are carried out by two separate enzymes,each containing its own binding site for the methyl donor, SAM.

Many viruses encode their own mRNA cap methylaion machinery, the best-studied exam‐ple of which is the poxvirus vaccinia virus. For vaccinia virus, the G-N-7 and 2’-O MTase activities are encoded by two separate viral proteins, D12L and VP39 [65, 68-70]. In the case of reovirus, G-N-7 and 2’-O MTases are catalyzed by two separate domains of the same viral polymerase protein [55, 71]. For VSV, G-N-7 and 2’-O MTases are accomplished by a single region (CR VI) located in the C terminus of viral polymerase protein, L (Fig.4C) [14, 17, 19].Soon after the discovery of the dual MTase activities of VSV, the N terminus of flaviviruses polymerase protein (NS5) was found to encode both G-N-7 and 2’-O MTases (Fig.4B)[72-74]. In addition to this unusual dual MTase activity of CR VI, the order of mRNA cap methylation in VSV is unconventional in which 2’-O methylation precedes and facilitates the G-N-7 methylation [17, 18]. This is contrast to all known mRNA cap methylation reactions including flaviviruses.

5.2. A single MTase catalytic site in CR-VI of L protein essential for both G-N-7 and 2’-O methylation

The SAM-dependent MTase superfamily contains a series of conserved motifs (X and I to VIII) [75]. The crystal structure of several known 2’-O MTases including E. coli heat shock-induced methyltransferase RrmJ/FtsJ and vaccinia virus VP39 have been solved[67, 68, 70,76]. In RrmJ, a catalytic tetrad of residues: K38, D124, K164, and E199 formed the active site of 2’-O MTase [67, 76]. Site-directed mutagenesis of RrmJ found that a catalytic triad of resi‐dues K38, D124, and K164 are essential for 2’-O MTase whereas E199 plays only a minor role in the methyltransferase reaction in vitro . In vaccinia virus VP39, four amino acids, K41,D138, K175, and E207, are essential for catalysis [68, 70]. By comparing the amino acid se‐quence of the RrmJ and VP39 with CR VI of the L protein of NNS RNA viruses, it was sug‐gested that this region of L protein might function as a 2′-O MTase. Sequence alignments suggest that residues K1651, D1762, K1795, and E1833 of the VSV L protein correspond to a catalytic KDKE tetrad (Fig.5). In fact, this KDKE motif is conserved in CR VI of L proteins of all NNS RNA viruses with the exception of Borna disease virus. Li et al., (2005) performed an extensive mutagenesis analysis in this predicted MTase catalytic KDKE tetrad in VSV L protein [14]. Recombinant VSVs carrying individual substitutions to K1651, D1762, K1795,and E1833 were recovered from an infectious cDNA clone of VSV. Analysis of the cap struc‐ture of mRNA synthesized in vitro revealed that alterations to the predicted active site resi‐dues abolished both G-N-7- and ribose 2′-O MTase activities. This result demonstrated that a single KDKE tetrad in CR-VI of the VSV L protein is essential for mRNA cap G-N-7- and Messenger RNA Cap Methylation in Vesicular Stomatitis Virus, a Prototype of Non‐Segmented Negative‐Sense RNA

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ribose 2′-O methylation [14]. Two models have been proposed to explain this result. One possibility is that CR VI functions as both G-N-7 and 2’-O MTases. However, this conflict the fact that all known G-N-7 and 2’-O MTases have distinct biochemistry during RNA methyl‐ation reactions. An alternative explanation is that there is a sequential model for VSV mRNA cap methylation in which the product of one MTase acts as the substrate for the sec‐

ond (discussed below).

Figure 5. Structure-based amino acid sequence alignments of conserved domain VI of representative NNS RNA virus L proteins with known 2'-O methyltransferase, the E. coli RrmJ and vaccinia virus VP39. The conserved motifs (X and I to VIII) correspond to the SAM-dependent MTase superfamily. MTase catalytic site is shown by pink color. SAM binding site are shown by yellow color. Conserved aromatic residues are shown by blue color. Predicted alpha-helical regions are shown by the cylinders and the ?-sheet regions by the arrows. STR, structure of RrmJ and predicted structure for the NNS RNA viruses; EBOM, Ebola virus; VSIV, VSV Indiana type; HRSV, human respiratory syncytial virus; RRMJ, E. coli heat shock 2'-O MTase; VP39, vaccinia virus 2'-O MTase VP39.

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5.3. A single SAM binding site in L protein essential for both G-N-7 and 2’-O methylation The SAM-dependent MTase superfamily usually contains a G-rich motif (GxGxG) and an acidic residue (D/E) that is involved in SAM binding [75]. Sequence alignments between CR VI of NNS RNA virus L proteins and known MTases suggest that the SAM-binding residues of VSV L include G1670, G1672, G1674, G1675, and D1735 (Fig.5). Site-directed mutagenesis has been performed to define the roles of these amino acids in VSV mRNA methylation [17]. Each of these residues was individually substituted for alanine (A); or,for G4A, all four G residues were replaced with A; for G4AD, residue D1735 was also replaced with A. In addition, the flanking amino acid residues D1671 and S1673 within GDGSG motif were also substituted. Recombinant viruses were recovered from each of the L gene mutations. It was found that mutations to G1670, G1672, and S1673 specifical‐ly diminished G-N-7, but not 2’-O methylation, suggesting that 2’-O methylation occurs prior to G-N-7 methylation in VSV [17]. In contrast, mutants D1671, G4A, G4AD,G1675A, and D1735 were defective in both 2'-O and G-N-7 methylations. Interestingly,mutant G1674A requires a higher concentration of SAM to achieve full methylation com‐pared with wild type VSV and methylation is more sensitive to SAH inhibition. There‐fore, amino acid substitutions to the predicted SAM binding site disrupted methylation at the G-N-7 position or at both the G-N-7 and ribose 2′-O positions of the mRNA cap.However, none of these mutants are specifically defective in 2’-O methylation alone.These studies provide genetic evidence that the two methylase activities share one single SAM binding site and, in contrast to other cap methylation reactions, methylation of the G-N-7 position is not required for 2′-O methylation.

5.4. Mapping the potential RNA binding site that required for mRNA cap methylation

To acquire methylation, the MTase usually directly or indirectly contacts an RNA sub‐strate. This putative substrate binding site is poorly understood in NNS RNA viruses.However, this substrate binding site has been identified in several cellular and viral mRNA 2'-O MTases [67, 68, 70, 74, 77, 78]. To achieve 2'-O methylation, the RNA sub‐strate interacts with the cap recognition site which requires stacking between the base of the cap and aromatic rings from a MTase [76, 79, 80]. Vaccinia VP39 is one of the best characterized 2'-O MTases. In VP39, it was found that the recognition of a methylated base is achieved by stacking between two aromatic residues (Y22 and F180) and the methyl group is in contact with residue Y204 (Fig.6A) [68, 70, 79]. In addition, the car‐boxyl groups of residues D182 and E233 form hydrogen bonds with the NH and NH2 of the guanosine in VP39 (Fig.6A). Based on structure modeling and mutagenesis analysis,it was shown that residue F24 in West Nile virus (WNV) methylase (NS5) [81, 82] and Y29 and F173 in feline coronavirus 2'-O MTase (nsp16) [80] may play an equivalent role to residue Y22 in VP39 of vaccinia virus. The cellular cap binding protein-eukaryotic translation initiation factor 4E (eIF-4E) recognizes the cap by stacking between W56 and W102 [83]. In all known cases, aromatic residues are involved in cap binding and sub‐strate recognition.

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Figure 6. The predicted structure of CR VI of VSV L protein . (A) Cap binding site of vaccinia virus 2'-O MTase,VP39. The model was generated from the crystal structure of VP39 (PDB code: 1AV6) using PyMOL. The side chains of amino acid residues (Y22, F180, E233 and D182) involved in binding of 7m Gp cap are shown as sticks. (B) The predict‐ed structure of CR VI of VSV L protein . The model was generated based on the previous predicted structure of VSV MTase (amino acid residue from 1644 to 1842 in L protein) using PyMOL software. Alpha helices are shown in blue and beta strands are shown in pink. SAM and the side chains of critical residues are shown in sticks, and the oxygen atoms and nitrogen atoms are shown in red and blue, respectively. For SAM molecule, the carbon atoms are green.For the three important amino acids (Y1650, F1691 and E1764) that may be involved in RNA substrate binding, their side-chain carbon atoms are highlighted in orange. For the predicted catalytic residues (K1651, D1762, K1795 and E1833), the carbon atoms are shown in purple.

Guided by this information, the putative RNA binding site in VSV L protein was searched through mutagenesis analysis of selected conserved residues in region VI of VSV L protein that were physiochemically similar to those involved in substrate recognition in VP39. Se‐quence alignment showed that there are a number of aromatic residues that are highly con‐served in the MTase domain of L proteins of NNS RNA viruses (Fig. 5). Aromatic residues at positions 1650 (Y), 1691(F or Y), and 1835 (Y) are highly conserved in L proteins of NNS RNA viruses. Aromatic residues at positions 1742 (W), 1744 (Y), 1745 (F), and 1816 (F) are conserved in the L proteins of Rhabdoviridae and some Paramyxoviridae and Filoviridae . There‐fore, these aromatic residues were selected as putative equivalents of Y22, F180, and Y204 in VP39. However, there is no amino acid precisely aligned with D182 and E233 in VP39. With the exception of two acidic amino acids in the catalytic site (K1651-D1762-K1795-E1833), po‐sition E1764 is also conserved in all L proteins. Thus, E1764 was selected as a candidate for mimicking the role of VP39 residues D182 and E233. In addition, two serine mutations at the two most conserved positions at 1693 and 1827 of VSV L protein was also examined, based on the fact that it has been shown that a serine residue was involved in RNA-protein interac‐tion in E. coli 2'-O MTase, RRMJ [67, 77]. To determine the role of these amino acid residues in mRNA cap methylation, a single point mutation was introduced to an infectious clone of VSV and recombinant VSVs harboring these mutations were recovered [84]. The importance of the maintenance of the aromatic ring at amino acids Y1650 and F1691 was revealed by the observation that the substitution of Y1650 and F1691 with two other possible aromatic resi‐dues in the VSV infectious clone still produced viable recombinant viruses and produced a fully methylated mRNA cap, but alanine substitutions dramatically inhibited viral replica‐Methylation - From DNA, RNA and Histones to Diseases and Treatment

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tion and completely blocked both G-N-7 and 2’-O methylation [84]. Based on the predicted structural model for the VSV MTase (amino acid residues from 1644 to 1842 in the L protein)(Fig.6B), the residues Y1650 and F1691 are located far from each other with a distance of 17.3? between their alpha carbon atoms. Y1650 is located in the middle of the first helix, and the F1691 is at the very C-terminal of the second helix. Perhaps, a stacking interaction with one aromatic residue causes a conformational and structural change in the VSV methylase,which results in the interaction with another aromatic residue. Changing of residue E1764 to D (maintenance of charge), Q (maintenance of size), or K (changing charge), even the very conservative change to D, dramatically inhibited both G-N-7 and 2'-O methylation [84]. The predicted structure of VSV MTase also shows that E1764, the residue adjacent to the catalyt‐ic residue D1762, is exposed to the putative SAM binding site (Fig.6B). The side chain of E1764 shows close contact to the adenyl group of SAM (3.1 ?). In addition, it was found that Y1835A was found to require a higher SAM concentration to achieve full methylation and it is more sensitive to MTase inhibitor [84].

To date, this work is the first attempt toward elucidation of the putative RNA substrate recogni‐tion site in the L protein of NNS RNA viruses, which has shed light on the possible role of several conserved aromatic amino acids, including Y1650 and F1691, in RNA binding during cap meth‐ylation. It would provide much more direct evidence for the role of these key amino acids in me‐diating RNA binding if the RNA binding efficiency could be measured directly. Attempts to use a gel shift assay have failed to this end [84], as the existence of multiple RNA binding sites in L protein with a size as large as 241-kDa posed a tremendous challenge in discerning the effect of single point mutation. The use of a truncated CR VI of VSV L for in vitro RNA binding assays might be a useful alternative strategy for future studies.

5.5. An unusual order for mRNA cap methylation in VSV

For conventional mRNA cap methylation, two separate MTases sequentially methylated the cap structure, first at the G-N-7 position and subsequently at the ribose 2′-O position [65, 66].Analysis of the cap methylation of mRNA synthesized in vitro suggests that mRNA cap methyla‐tion in VSV is unusual, with methylation of ribose 2′-O occurring prior to G-N-7 methylation.First, early studies showed that at low concentrations of SAM, VSV mRNA was methylated at the 2’-O position only [85]. However, it could be chased into a doubly methylated cap structure at high SAM concentrations in vitro . Second, when in vitro mRNA synthesis was performed in the presence of MTase inhibitors such as SAH and sinefungin, G-N-7 methylation was inhibited prior to 2’-O methylation [86]. Third, a host range mutant of VSV, hr 8, was shown to synthesize mRNA cap structures that lacked G-N-7 but were partially 2′-O-methylated [46, 87]. Finally,VSV mutants carrying mutations in the SAM binding site (such as G1670A, and G1672A) are spe‐cifically defective in G-N-7, but not 2’-O methylation [17].

This unusual order of VSV mRNA cap methylation was also biochemically demonstrated by a trans -methylation assay in which both ribose 2′-O and G-N-7 MTases were recapitulated by using purified recombinant L and in vitro -synthesized RNA [18]. It was found that VSV L modifies the 2′-O position of the cap prior to the G-N-7 position and that G-N-7 methylation is diminished by pre-2′-O methylation of the substrate RNA [18], providing compelling evi‐Messenger RNA Cap Methylation in Vesicular Stomatitis Virus, a Prototype of Non‐Segmented Negative‐Sense RNA

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dence that 2′-O methylation precedes and facilitates G-N-7 methylation. In light that both two MTase activities appear to reside in the same domain of L protein with the same SAM binding site, it is conceivable that mRNA cap and/or the SAM binding site might need to be repositioned at the end of the first methylation reaction to facilitate the second round of methylation. How this coordination happens in vitro is still a mystery. Bearing in mind that G-N-7 position is upstream of the ribose 2'-O position in the mRNA strand, reorientation is thus less likely to have resulted from forward movement of capped RNA through CR VI during transcription, but rather it might entail a fine spatial rearrangement. Collectively,these experiments have shown that the order of VSV mRNA cap methylation is distinct from all other known mRNA cap methylation mechanisms.

5.6. VSV methylases require cis -element in RNA

During mRNA synthesis, the VSV polymerase initiates synthesis at the first gene-start (GS)sequence (3′ UUGUCNNUAC 5′), and the nascent mRNA chain is capped and methylated,and recognizes a specific gene-end (GE) sequence (3′-AUACUUUUUUU-5′), the polymerase polyadenylates and terminates. It has been well demonstrated that the GS sequence contains a key cis -acting regulatory element for the initiation of mRNA synthesis [31, 32]. Specifically,the first three positions of the GS sequence have been found to be critical for mRNA synthe‐sis. Recently, both trans capping assays with 5-nt oligo RNA substrates and detergent-acti‐vated virus transcription reactions pointed out the importance of positions 1, 2, 3, and 5 in mRNA cap addition, although position 5 substitutions were more tolerated [12, 31, 32]. Us‐ing a trans methylation assay, it was found that similar signals were required for mRNA cap methylation [18]. As expected, VSV L protein efficiently methylated a 110 nt of RNA with an authentic gene start sequence at position 2’-O. However, when the gene start sequence of this 110 nt was replaced with non-viral sequence (5′ GpppGGACGAAGAC-RNA), the effi‐ciency of 2’-O methylation was reduced approximately 9 times. Similarly, VSV L protein ef‐ficiently methylated a pre-2′-O-methylated VSV mRNA at position G-N-7. In contrast, the efficiency of G-N-7 methylation decreased nearly 7 times when incubated with a substrate with non-VSV mRNA start RNA. Therefore, the gene start sequence of VSV mRNA contains a signal for initiation of mRNA synthesis, mRNA cap addition, and cap methylation.

5.7. The length of mRNA in cap methylation

In the trans mRNA capping assay, the VSV L protein efficiently caps the 5-nt gene start se‐quence [12, 16], demonstrating that a 5-nt RNA substrate is sufficient for mRNA cap addi‐tion. In order to determine the minimum length of RNA required for mRNA cap methylation, 5-,10-, 51-, and 110-nt RNAs were used as substrates for a trans methylation as‐say in vitro [18]. Interestingly, the 10-, 51-, and 110-nt RNAs were able to serve as substrates for both G-N-7 and ribose 2'-O methylations, whereas the 5-nt RNA was not methylated by the VSV L protein at either the G-N-7 or the ribose 2'-O position [18]. Therefore, in contrast to trans capping, a 5-nt substrate is not sufficient for trans methylation and likely the con‐served positions 8, 9, and 10 in VSV gene start sequence are required for mRNA cap methyl‐ation. Clearly, the length of RNA required for methylation is longer than that required for capping by the VSV L protein.

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5.8. Model for mRNA cap methylation

The process of VSV L protein-mediated cap methylation can be best summarized with the following model (Fig.7). Initially in response to a specific cis-acting element in the VSV gene start sequence, CR VI of L protein methylates the cap structure first at the 2'-O position to produce GpppA m ACAG-RNA. The by-product of this reaction, SAH, is released from this reaction. Following 2’-O methylation, a second molecule of SAM binds to CR VI of L protein that may facilitate a subsequent methylation of the RNA at the G-N-7 position. Methylation at the 2'-O position favors G-N-7 methylation in the cap structure through a currently un‐known mechanism. G-N-7 methlyation may be facilitated by the contact with the RNA mol‐ecule remains bound to the L protein at the end of the initial methylation at the 2'-O position. Another possibility is that CR VI of L protein exists in two different conformations.An initial conformation may favor binding of GpppRNA and SAM. Methylation of RNA at the 2'-O position perhaps induces a conformational change that facilitates the repositioning of the RNA for subsequent G-N-7 methylation, and/or favors the release of SAH as well as the binding of a subsequent molecule of SAM.

5.9. Comparison of mRNA cap methylation in VSV and WNV

To date, the rhabdovirus, VSV, and the flavivirus, WNV, are the two best characterized vi‐ruses that utilize a single region in the polymerase protein for both G-N-7 and 2'-O methyla‐tions. However, the mechanism of VSV mRNA methylation is distinct from that of the WNV system (Fig.4B and C). In VSV, 2'-O methylation precedes and facilitates subsequent G-N-7methylation [17, 18]. However, WNV MTases modify the cap structure, first at the G-N-7position and subsequently at the ribose 2'-O position [72, 73, 78]. In VSV, the G-N-7 and 2'-O MTases require similar conditions for methylation with an optimal pH at 7.0 [18]. In con‐trast, the G-N-7 and 2'-O MTases of WNV require an optimal pH at 6.5 and 10, respectively [72]. Both VSV and WNV MTases modify the RNA in a sequence-specific manner, but re‐quire different elements in the RNA substrate. VSV G-N-7 and 2'-O MTases require specific gene start sequences with a minimum mRNA length of 10 nucleotides [18]. In the WNV model, N-7 cap methylation requires the presence of specific nucleotides at the second and third positions and a 5' stem-loop structure within the 74-nucleotide viral RNA; in contrast,2'-O ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5' viral RNA of 20 nucleotides in length [81]. In addition, there is striking differ‐ence in the cap recognition site between the VSV and WNV MTases. For the WNV MTase,the cap recognition site is essential for 2'-O, but not G-N-7 methylation [73, 82]. Consistent with this finding, it was found that GTP and cap analogs specifically inhibited 2'-O, but not G-N-7 methylation [73, 82]. However, mutations to the putative RNA binding site in VSV L protein affected both G-N-7 and 2’-O methylations. GTP and cap analogs did not affect VSV mRNA cap methylation in vitro . Overall, the mechanism of VSV mRNA cap methylation is significantly different from that of WNV. Most recently, it was found that capping of flavivi‐rus RNA is catalyzed by conventional RNA guanylyltransferase via a covalent GMP-en‐zyme intermediate [88]. However, VSV capping is catalyzed by a novel PRNTase [12, 13, 16,89]. These studies suggest that VSV and perhaps other NNS RNA viruses have evolved a unique mechanism to add the cap to their mRNA and to methylate the cap structure.

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Figure 7. Proposed model for mRNA cap methylation in VSV and eukaryotic cells. (A) VSV MTase model. VSV mRNA cap structure is first methylated by CR VI of L protein at the 2'-O position to produce GpppA m ACAG-RNA. The by-product, SAH, is released before the binding of a second molecule of SAM. G-N-7 methlyation may be facilitated by the contact with the RNA molecule remains bound to the polymerase at the end of the initial methylation at the 2'-O position. Or, methylation of RNA at the 2'-O position may induce a conformational change that facilitates the reposi‐tioning of the RNA for subsequent G-N-7 methylation. (B) Cellular MTase model. GpppNNNN-RNA is first methylated by a G-N-7 MTase to yield 7m GpppNNNN-RNA. Following G-N-7 methylation, 7m GpppNNNN-RNA dissociates with G-N-7 MTase, and re-associates with a separate 2’-O MTase to yield 7m GpppN m NNN-RNA. In this model, the two methyl‐ase activities are carried out by two separate enzymes, each containing its own SAM binding site.

5.10. mRNA cap methylation in other NNS RNA viruses

Limited accomplishments have been made in understanding the mechanism of mRNA cap methylation in other NNS RNA viruses, due to the lack of a robust in vitro mRNA synthesis for most of NNS RNA viruses, and the technical challenge of expression and purification of a func‐tional polymerase protein or fragment. In early 1970, Colonno and Stone showed that the NDV mRNA cap structure was methylated only at G-N-7 [90]. This is distinct from the cap structures of other NNS RNA viruses which typically contain two methyl groups, at positions G-N-7 and ribose 2'-O. However, detailed characterization of the NDV methylase activities and the mech‐anism involved in this unique methylation is not understood. The mechanism underlying this difference and the biological significance of the lack of 2’-O MTase is not known. Sequence analysis has revealed that the proposed SAM binding region for the Filoviridae , Rubulavirus ,and Avulavirus genera of the Paramyxoviridae contains a conserved AxGxG sequence rather than GxGxG within motif I of the SAM-dependent MTase superfamily (Fig.8). It will be inter‐esting to determine if there is a link between this differential SAM binding sites and the lack of 2'-O methylation in NDV. Recently, it was shown that a fragment of Sendai virus L protein that includes CR VI was able to methylate short Sendai virus-specific RNA sequences in trans at the Methylation - From DNA, RNA and Histones to Diseases and Treatment

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G-N-7 position [43]. However, the "trans -methylation" assay used in their study does not allow detection of 2’-O methylation although it is known that Sendai virus encodes two MTases.More recently, recombinant Sendai virus carrying mutations in catalytic KDKE tetrad and SAM binding site were recovered from infectious clones [91, 92]. It was found that these muta‐tions affected mRNA cap methylation. However, whether they are specifically defective in G-N-7 and/or 2’-O methylation is not known because of the assay did not have the ability to distinguish the two methyl groups. In addition, the order of methylation may be different among NNS RNA viruses. In contrast to VSV, methylation of RSV mRNA at low SAM concen‐trations was found at only the G-N-7 position; however, it was found to be doubly methylated at high SAM concentrations [63]. This study provided evidence that the order of mRNA cap methylation in RSV may be different with VSV. Clearly, more studies are needed to under‐

stand the mRNA cap methylation in other NNS RNA viruses.

Figure 8. SAM binding motifs in NNS RNA virus MTases. The proposed SAM binding region for the Filoviridae , Ru‐bulavirus , and Avulavirus genera of the Paramyxoviridae contains a conserved AxGxG sequence rather than GxGxG.NNS RNA viruses include VSVI, VSV Indiana; VSVN, VSV New Jersey; BEFV, bovine emphemeral fever virus; RABV, rabies virus; Marb, Marburg virus; MeV: measles; HV, Hendra virus; NPV, Nipah virus; AMPV, avian metapneuomovirus;HMPV, human metapneuomovirus; APMV6, avian paramyxovirus 6; MuV, mumps virus; HPIV2, human parainfluenza virus 2; SV5, simian virus 5; SV41, simian virus 41;

SS(+) RNA: single strand positive RNA viruses include DNV, dengue virus; WNV, West Nile virus; YFV, yellow fever virus.

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6. The effects of 5’ mRNA cap addition and cap methylation on 3’ mRNA polyadenylation

VSV mRNA is capped and methylated at the 5′ end and polyadenylated at the 3′ end.Cap addition, cap methylation, and polyadenylation are carried out by three different re‐gions (CR V, CR VI, and CR III) in L protein. During VSV mRNA synthesis, modifica‐tions of the 5′ and 3′ ends of the mRNAs are tightly coupled to transcription [21-23].Although the detailed mechanism by which the polymerase coordinates these modifica‐tion events is poorly understand, available evidence suggests there is a link between au‐thentic 5′-end formation and 3′-end formation during VSV mRNA synthesis. Early studies demonstrated that the length of poly (A) tails on VSV mRNAs is affected by the presence of SAH, the by-product and competitive inhibitor of SAM-mediated methyl‐transferases [93-95]. The fact that the polymerase can synthesize full-length mRNAs in vi‐tro in the absence of SAM or in the presence of SAH, suggests that transcription is not dependent on cap methylation. When in vitro transcription reactions are performed in the presence of SAM, the RdRp synthesizes mRNA with poly (A) tail of 100 to 200 nt in length, similar to those synthesized in VSV-infected cells. However, when VSV mRNA cap methylation was inhibited during in vitro transcription reactions by supplementing with 1 mM SAH, the synthesized mRNA was heterogeneous in length due to having ex‐tremely long poly (A) tails, from 700 to 2,400 nucleotides (nt) in length [93, 96]. Recent work demonstrated that SAH-induced hyperpolyadenylation also occurs in cells infected by wild-type VSV in the presence of adenosine dialdehyde (AdOX), a compound that in‐hibits the activity of SAH hydrolase [97]. These results indicated that chemical inhibition of VSV mRNA cap methylation by SAH resulted in hyperpolyadenylation of viral mRNA. Interestingly, this hyperpolyadenylation of VSV mRNAs has been observed in ts (G)16, a VSV mutant identified in 1970 based on its ability to grow at 31°C but not at 39°C [93, 98, 99]. In the absence of SAH, the mRNAs synthesized by VSV mutant ts (G)16were hyperpolyadenylated at the 3′ end. Genomic sequence analysis found that the L protein of ts (G)16 contains two amino acid changes, C1291Y and F1488S, compared to wild type. Combined with the analysis of revertants of ts (G)16, it was found that F1488S,located in the variable region of L between CR V and CR VI, is responsible for the hy‐perpolyadenylating phenotype.

The characterization of a panel of MTase-defective VSVs may serve as a tool to understand the mechanism by which SAH or the failure to methylate the cap structure results in hyper‐polyadenylation. It was found that rVSV-K1651A, a mutation in MTase active site and com‐pletely defective in G-N-7 and 2’-O methylation, synthesized excessively long poly(A) tails,similar to those produced by wild-type L in the presence of SAH [15]. Similarly, the substi‐tution D1762E at the MTase active site, which inhibits both G-N-7 and 2’-O methylation,produces large polyadenylate in the presence or absence of SAH [97]. This data confirms the earlier work demonstrating that the inhibition of cap methylation results in large polyade‐nylate. In contrast, several other substitutions that inhibit cap methylation, including D1762G, D1762N, G1672P, and G1675P, did not produce hyperpolyadenylated mRNA [97].Methylation - From DNA, RNA and Histones to Diseases and Treatment

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Perhaps, K 1651A and D1762E substitutions might favor the binding of SAH at the SAM binding site in CR VI, resulting in hyperpolyadenylation without the need for supplemental SAH. Clearly, further studies are needed to understand the relationship between 5’ mRNA cap methyaltion and 3’ polyadenylation.

However, it appears clear that 5’ cap addition is required for 3’ polyadenylation, as evi‐denced by the polymerase mutants (G1154, T1157, H1227, and R1228) within CR V of L that inhibited cap addition also inhibit polyadenylation [15]. These cap-defective polymerases synthesized truncated transcripts that predominantly terminated within the first 500 nt of the N gene and contained short A-rich sequences at their 3′ termini. To examine how the cap-defective polymerases respond to an authentic VSV termination and re-initiation signal present at each gene junction, a 382 nt gene was inserted at the leader-N gene junction in the VSV genome. Using this N-RNA as the template, the cap-defective polymerases were able to synthesize full-length 382-nt transcripts that were not capped at 5’end. Interestingly, these uncapped transcripts lacked an authentic polyadenylate tail and instead contained 0 to 24 A residues [15]. In addition, the cap-defective polymerases were also unable to efficiently copy the downstream genes [15]. This finding strongly supports that 5′ mRNA cap addition and 3′ polyadenylation are mechanistically and functionally linked.

7. Impact of mRNA cap methylation on viral replication and gene

expression

In eukaryotic cells, it is well established that G-N-7 methylation of the mRNA cap structure is essential for mRNA stability and efficient translation [11, 48, 66]. Specifically, G-N-7 meth‐ylation of the mRNA cap structure is required for recognition of the cap by the rate limiting factor for translation initiation, eIF-4E [100, 101]. The mRNA cap structures of NNS RNA vi‐ruses are typically G-N-7 and 2’-O methylated. Although the precise mechanism by which VSV mRNAs are translated is unclear, they are broadly thought to utilize a variation of the canonical cap-dependent translational pathway [102-104]. In VSV-infected cells, host mRNA translation is rapidly inhibited through the suppression of the intracellular pools of eIF-4E by a manipulation of the phosphorylation status of the 4E binding protein (4E-BP1) [104].Nevertheless, in vitro experiments have shown that G-N-7 cap methylation facilitates trans‐lation of VSV proteins. MTase-defective VSV would provide a tool to study the role of mRNA cap methylation in viral protein synthesis. Ultimately, it will affect viral genome rep‐lication and gene expression since viral replication requires ongoing protein synthesis.

Based on the status of mRNA methylation, MTase-defective VSVs can be classified into three groups [14, 17, 84]. Viruses in the first group are completely defective in both G-N-7and 2’-O methylation, including mutations in MTase active site (rVSV-K1651A, D1762A,K1795A, E1833Q, and E1833A), SAM binding site (rVSV-D1671V, G1675A, G4A, and G4AD), and putative RNA binding site (rVSV-Y1650A, F1691A, and E1764A). Viruses in the second group are specifically defective in G-N-7, but not 2’-O MTase, including mutants in SAM binding site (rVSV-G1670A, G1672A, and S1673A). Viruses in third group that require Messenger RNA Cap Methylation in Vesicular Stomatitis Virus, a Prototype of Non‐Segmented Negative‐Sense RNA

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elevated SAM concentrations to permit full methylation including a mutant in SAM binding site (rVSV-G1674A) and putative RNA binding site (rVSV-Y1835A). With the exception of rVSV-G1674A and Y1835A, all MTase-defective VSVs were attenuated in cell culture as judged by diminished viral plaque size, reduced infectious viral progeny release (in single-step growth curves), and decreased levels of viral genomic RNA, mRNA, and protein syn‐thesis. It appears that the degree of attenuation is consistent with the defects of the methylation. For example, viruses defective in both G-N-7 and 2’-O methylation had 2-5 log reductions in growth whereas viruses only defective in G-N-7 had 1-2 log declines in repli‐cation [14]. Recombinant rVSV-G1674A and Y1835A replicated as efficiently as wild type rVSV [14]. A remarkable finding is that some of the mutants in the SAM binding site (rVSV-G1675A, G4A, and G4AD) affected transcription and replication differently [17]. For these mutants, replication was enhanced 2.5- to 4-fold, and transcription decreased up to 8-fold compared with rVSV. One feature of the gene expression strategy of NNS RNA viruses is that the polymerase complex controls two distinct RNA synthetic events: genomic RNA rep‐lication and mRNA transcription [20, 105, 106]. It is possible that SAM binding influences the switch of polymerase between replicase and transcriptase. Perhaps, L protein with SAM binding favors to function as transcriptase, whereas L protein that lacks SAM binding favors replicase function.

8. Impact of mRNA cap methylation on viral pathogenesis in vitro

Although it is well studied that MTase-defective viruses were attenuated in cell culture, the impact of mRNA cap methylation on viral pathogenesis in vitro is poorly understood. Re‐cently, Ma et al., (2012) examined the pathogenicity of MTase-defective VSVs in mice [107].VSV infects a wide range of wild and domestic animals such as cattle, horses, deer, and pigs,characterized by vesicular lesions in the mouth, tongue, lips, gums, teats, and feet. Although the mouse is not the natural host of VSV, it represents an excellent small animal model to understand VSV pathogenesis because VSV causes systemic infection and fatal encephalitis [108-110]. After intranasal inoculation, VSV infects olfactory neurons in the nasal mucosa and subsequently enters the central nervous system (CNS) through the olfactory nerves. The virus is then disseminated to other areas in the brain through retrograde and possibly ante‐rograde trans-neuronal transport, ultimately causing an acute brain infection. It was found that VSV mutants, rVSV-K1651A, D1762A, and E1833Q, which have mutations in the MTase catalytic site and are defective in both G-N-7 and 2’O methylation, were highly attenuated in mice [107]. Mice inoculated with these recombinant viruses did not show any clinical signs of VSV infection such as weight loss, ruffled fur, hyperexcitability, tremors, circling, and pa‐ralysis. Furthermore, these mutant viruses were not able to enter the brain, had dramatic de‐fects in replication in lungs, and did not cause significant histopathological changes in lungs and brain. Recombinant rVSV-G1670A and G1672A, which have mutations in the SAM binding site and are defective in G-N-7 but not 2’-O methylation, retained low virulence in mice [107]. Mice inoculated these two recombinants exhibited weight loss of approximately 2-3 g during days 3-7 post-inoculation and showed mild illnesses such as ruffled coat for 2-3Methylation - From DNA, RNA and Histones to Diseases and Treatment

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刘祖洞遗传学第三版标准答案第9章数量性状遗传

第九章数量性状遗传 1.数量性状在遗传上有些什么特点？在实践上有什么特点？数量性状遗传和质量性状遗传有什么主要区别？解析：结合数量性状的概念和特征以及多基因假说来回答。参考答案：数量性状在遗传上的特点：（1）数量性状受多基因支配（2）这些基因对表型影响小，相互独立，但以积累的方式影响相同的表型。（3）每对基因常表现为不完全显性，按孟德尔法则分离。数量性状在实践上的特点：（1）数量性状的变异是连续的，比较容易受环境条件的影响而发生变异。（2）两个纯合亲本杂交，F1 表现型一般呈现双亲的中间型，但有时可能倾向于其中的一个亲本。F2的表现型平均值大体上与F1 相近，但变异幅度远远超过F1。F2 分离群体内，各种不同的表现型之间，没有显着的差别，因而不能得出简单的比例，因此只能用统计方法分析。（3）有可能出现超亲遗传。数量性状遗传和质量性状遗传的主要区别：（1）数量性状是表现连续变异的性状，而质量性状是表现不连续变异的性状；（2）数量性状的遗传方式要比质量性状的遗传方式复杂的多，它是由许多基因控制的，而且它们的表现容易受环境条件变化的影响。 2.什么叫遗传率？广义遗传率？狭义遗传率？平均显性程度？解析：根据定义回答就可以了。参考答案：遗传率指亲代传递其遗传特性的能力，是用来测量一个群体内某一性状由遗传因素引起的变异在表现型变异中所占的百分率，即：遗传方差/总方差的比值。广义遗传率是指表型方差（Vp ）中遗传方差（Ve）所占的比率。狭义遗传率是指表型方差（V p ）中加性方差（V A）所占的比率。平均显性程度是指V D /V A 。〔在数量性状的遗传分析中，对于单位点模型，可以用显性效应和加性效应的比值d/a 来表示显性程度。但是推广到多基因

刘祖洞遗传学课后题答案

第二章孟德尔定律 1、为什么分离现象比显、隐性现象有更重要的意义答：因为（1）分离规律是生物界普遍存在的一种遗传现象，而显性现象的表现是相对的、有条件的；（2）只有遗传因子的分离和重组，才能表现出性状的显隐性。可以说无分离现象的存在，也就无显性现象的发生。 9、真实遗传的紫茎、缺刻叶植株（AACC ）与真实遗传的绿茎、马铃薯叶植株（aacc ）杂交，F2结果如下：紫茎缺刻叶紫茎马铃薯叶绿茎缺刻叶绿茎马铃薯叶 247 90 83 34 （1）在总共454株F2中，计算4种表型的预期数。（2）进行2 测验。（3）问这两对基因是否是自由组合的紫茎缺刻叶紫茎马铃薯叶绿茎缺刻叶绿茎马铃薯叶观测值（O ） 247 90 83 34 预测值（e ） (四舍五入) 255 85 85 29 454 .129 )2934(85)85583(85)8590(255)255247()(2 22 222 =-+ -+-+ -=-=∑e e o χ 当df = 3时，查表求得：＜P ＜。这里也可以将与临界值81.72 05.0.3=χ比较。可见该杂交结果符合F 2的预期分离比，因此结论，这两对基因是自由组合的。 11、如果一个植株有4对显性基因是纯合的。另一植株有相应的4对隐性基因是纯合的，把这两个植株相互杂交，问F2中：（1）基因型，（2）表型全然象亲代父母本的各有多少解：(1) 上述杂交结果，F 1为4对基因的杂合体。于是，F2的类型和比例可以图示如下：也就是说，基因型象显性亲本和隐性亲本的各是1/28 。 (2) 因为，当一对基因的杂合子自交时，表型同于显性亲本的占3／4，象隐性亲本的占1／4。所以，当4对基因杂合的F 1自交时，象显性亲本的为(3/4)4 ，象隐性亲本的为(1/4)4 = 1/28 。第三章遗传的染色体学说

刘祖洞遗传学习题答案

第六章染色体和连锁群 1、在番茄中，圆形（O ）对长形（o ）是显性，单一花序（S ）对复状花序（s ）是显性。这两对基因是连锁的，现有一杂交得到下面4种植株：圆形、单一花序（OS ）23 长形、单一花序（oS ）83 圆形、复状花序（Os ）85 长形、复状花序（os ）19 问O —s 间的交换值是多少解：在这一杂交中，圆形、单一花序（OS ）和长形、复状花序（os ）为重组型，故O —s 间的交换值为：%20%10019 85832319 23=?++++= r 2、根据上一题求得的O —S 间的交换值，你预期杂交结果，下一代4种表型的比例如何解： O_S_ ：O_ss ：ooS_ ：ooss = 51% ：24% ：24% ：1%，即4种表型的比例为：圆形、单一花序(51％)，圆形、复状花序(24％)，长形、单一花序(24％)，长形、复状花序(1％)。 3、在家鸡中，白色由于隐性基因c 与o 的两者或任何一个处于纯合态有色要有两个显性基因C 与O 的同时存在，今有下列的交配： ♀CCoo 白色 × ♂ccOO 白色 ↓ 子一代有色子一代用双隐性个体ccoo 测交。做了很多这样的交配，得到的后代中，有色68只，白

色204只。问o —c 之间有连锁吗如有连锁，交换值是多少解：根据题意，上述交配： ♀ CCoo 白色 ccOO 白色 ♂ ↓ 有色CcOo ccoo 白色 ↓ 有色C_O_ 白色（O_cc ，ooC_，ccoo ） 416820468=+ 4 3 68204204=+ 此为自由组合时双杂合个体之测交分离比。可见，c —o 间无连锁。 (若有连锁，交换值应为50％，即被测交之F1形成Co ：cO ：CO ：co ＝1 ：1 ：1 ：1的配子；如果这样，那么c 与o 在连锁图上相距很远，一般依该二基因是不能直接测出重组图距来的)。 4、双杂合体产生的配子比例可以用测交来估算。现有一交配如下：问：（1）独立分配时，P= （2）完全连锁时，P= （3）有一定程度连锁时，p= 解：题目有误，改为：)2 1( )21 (aabb aaBb Aabb AaBb p p p p -- （1）独立分配时，P = 1/4；（2）完全连锁时，P = 0；（3）有一定程度连锁时，p = r /2，其中r 为重组值。 5、在家鸡中，px 和al 是引起阵发性痉挛和白化的伴性隐性基因。今有一双因子杂种公鸡 al Px Al px 与正常母鸡交配，孵出74只小鸡，其中16只是白化。假定小鸡没有一只早期死亡，而px 与al 之间的交换值是10％，那么在小鸡4周龄时，显出阵发性痉挛时，（1）在白化小鸡中有多少数目显出这种症状，（2）在非白化小鸡中有多少数目显出这种症状解：上述交配子代小鸡预期频率图示如下： ♀W Al Px al Px Al px ♂

刘祖洞遗传学习题答案13

第七章细菌和噬菌体的重组和连锁 1.为什么说细菌和病毒是遗传学研究的好材料？ 2.大肠杆菌的遗传物质的传递方式与具有典型减数分裂过程的生物有什么不同？ 3.解释下列名词：（1）F-菌株，F+菌株，Hfr菌株；（2）F因子，F，因子，质粒，附加体；（3）溶源性细菌，非溶源性细菌；（4）烈性噬菌体，温和噬菌体，原噬菌体；（5）部分合子（部分二倍体）； 4.部分合子在细菌的遗传分析中有什么用处？ 5.什么叫转导、普遍性转导、特异性转导（局限性转导）？ 6.转导和性转导有何不同？ 7.一个基因型为a+b+c+d+e+并对链霉素敏感的E.coliHfr菌株与基因型为a-b-c-d-e-并对链霉素耐性的F-菌株接合，30分钟后，用链霉素处理，然后从成活的受体中选出e+型的原养型，发现它们的其它野生型（+）基因频率如下：a+70％，b+-，c+85％，d+10％。问a，b，c，d 四个基因与供体染色体起点（最先进入F-受体之点）相对位置如何？解：根据中断杂交原理，就一对接合个体而言，某基因自供体进入受体的时间，决定于该基因同原点的距离。因此，就整个接合群体而论，在特定时间内，重组个体的频率反映着相应基因与原点的距离。报据题目给定的数据，a、b、c、d与供体染色体的距离应该是： 8.为了能在接合后检出重组子，必须要有一个可供选择用的供体标记基因，这样可以认出重组子。另一方面，在选择重组子的时候，为了不选择供体细胞本身，必须防止供体菌株的继续存在，换句话说，供体菌株也应带有一个特殊的标记，能使它自己不被选择。例如供体菌株是链霉素敏感的，这样当结合体（conjugants）在含有链霉素的培养基上生长时，供体菌株就被杀死了。现在要问：如果一个Hfr菌株是链霉素敏感的，你认为这个基因应位于染色体的那一端为好，是在起始端还是在末端？解：在起始端 9.有一个环境条件能使T偶数噬菌体（T-even phages）吸附到寄主细胞上，这个环境条件就是色氨酸的存在。这种噬菌体称为色氨酸需要型（C）。然而某些噬菌体突变成色氨酸非依

遗传学试题刘祖洞版

第一章绪论一、选择题： 1.涉及分析基因是如何从亲代传递给子代以及基因重组的遗传学分支是：( ) A) 分子遗传学B) 植物遗传学C) 传递遗传学D) 种群遗传学 2.被遗传学家作为研究对象的理想生物，应具有哪些特征？( ) A)相对较短的生命周期B)种群中的各个个体的遗传差异较大 C)每次交配产生大量的子代D)遗传背景较为熟悉E)以上均是理想的特征二、名词解释 1.遗传学： 2.遗传： 3.变异： 4.进化遗传学： 5.发育遗传学： 6.免疫遗传学： 7.细胞遗传学： 8.人类遗传学：三、问答题 1.简述遗传学研究的对象和研究的任务。 2.为什么说遗传、变异和选择是生物进化和新品种选育的三大因素？ 3. 为什么研究生物的遗传和变异必须联系环境？ 4.遗传学建立和开始发展始于哪一年，是如何建立？ 5.为什么遗传学能如此迅速地发展？ 6.简述遗传学对于生物科学、生产实践的指导作用。 7.什么是遗传学？主要研究内容是什么？ 8．遗传学研究的对象是什么？ 9．遗传学在工农业生产和医疗保健上有何作用？ 10．在遗传学发展中大致分为几个阶段？有那些人做出了重大贡献？ 11．写出下列科学家在遗传学上的主要贡献。（1）Mendel (2) Morgan (3) Muller (4) Beadle 和Tatum (5)Avery (6) Watson 和Crick (7)Chargaff (8) Crick (9) Monod 和Jacob 第二章孟德尔定律一、选择题 1、最早根据杂交实验的结果建立起遗传学基本原理的科学家是：( ) A) James D. Watson B) Barbara McClintock C) Aristotle D) Gregor Mendel 2、以下几种真核生物，遗传学家已广泛研究的包括：( ) A) 酵母B) 果蝇C) 玉米D) 以上选项均是 3、通过豌豆的杂交实验，孟德尔认为；( ) A) 亲代所观察到的性状与子代所观察到相同性状无任何关联 B) 性状的遗传是通过遗传因子的物质进行传递的 C) 遗传因子的组成是DNA D) 遗传因子的遗传仅来源于其中的一个亲本 E) A 和C 都正确 4、生物的一个基因具有两种不同的等位基因，被称为：( ) A) 均一体B) 杂合体C) 纯合体D) 异性体E) 异型体 5、生物的遗传组成被称为：( )

遗传学名解-刘祖洞版

遗传学名解刘祖洞版 1性状（character）：遗传学中把生物体所表现的形态特征和生理特征，统称为性状。 2单位性状（unit character）：孟德尔在研究豌豆等植物的性状遗传时，把植株所表现的性状总体区分为各个单位作为研究对象，这样区分开来的性状称为单位性状。 3相对性状（contrasting character）：不同个体在单位性状上常有着各种不同的表现，例如：豌豆花色有红花和白花、种子形状有圆粒和皱粒。遗传学中同一单位性状的相对差异，称为相对性状。 4基因型(genotype) ：个体的基因组合。基因型是性状表现必须具备的内在因素。 5表现型（phenotype）：植株所表现出来的红花和白花性状(形态)就是表现型。表现型是指生物体所表现的性状。它是基因型和外界环境作用下具体的表现，是可以直接观测的。而基因型是生物体内在的遗传基础，只能根据表现型用实验方法确定。 6纯合的基因型（homozygous genotype）：成对的基因都是一样的基因型。如CC或cc。也称纯合体（h omozygote）。 7杂合的基因型（heterozygous genotype），或称杂合体（heterozygote）：成对的基因不同。如Cc。 8随体（Satellite）是指次缢痕区至染色体末端的部分，有如染色体的小卫星。随体主要由异染色质组成，是高度重复的DNA序列。 9无丝分裂（amitosis）：细胞核拉长呈哑铃状分裂，中部缢缩形成2个相似的子细胞。分裂中无染色体和纺锤体形成。如：纤毛虫、原生生物、特化的动物组织。 10有丝分裂(mitosis)：即体细胞分裂，通过分裂产生同样染色体数目的子细胞。在分裂中出现纺锤体。 11无性生殖(asexual reproduction)：通过有丝分裂，从一共同的细胞或生物繁殖得到的基因型完全相同的细胞或生物。也即克隆（clone）。 12有性生殖(sexual reproduction)：减数分裂和受精有规则地交替进行，产生子代的生殖方式。 13无融合生殖（apomixis）不经过雌雄配子融合而能产生种子的一种生殖方式。无融合生殖的方式根据无融合生殖最后形成胚是由减数后单倍雌配子直接发育而成，还是由未减数二倍细胞产生的，可以将无融合生殖分成三大类一类是减数胚囊中的无融合生殖另一类是未减数胚囊中的无融合生殖再一类是不定胚生殖 14反应规范（reaction norm）：基因型决定着个体对这种或那种环境条件的反应。

刘祖洞遗传学习题答案

1、在番茄中，圆形（O ）对长形（o ）是显性，单一花序（S ）对复状花序（s ）是显性。这两对基因是连锁的，现有一杂交得到下面4种植株：圆形、单一花序（OS ）23 长形、单一花序（oS ）83 圆形、复状花序（Os ）85 长形、复状花序（os ）19 问O —s 间的交换值是多少？解：在这一杂交中，圆形、单一花序（OS ）和长形、复状花序（os ）为重组型，故O —s 间的交换值为：%20%10019 85832319 23=?++++= r 2、根据上一题求得的O —S 间的交换值，你预期杂交结果，下一代4种表型的比例如何？ O_S_ ：O_ss ：ooS_ ：ooss = 51% ：24% ：24% ：1%，即4种表型的比例为：圆形、单一花序(51％)，圆形、复状花序(24％)，长形、单一花序(24％)，长形、复状花序(1％)。 3、在家鸡中，白色由于隐性基因c 与o 的两者或任何一个处于纯合态有色要有两个显性基因C 与O 的同时存在，今有下列的交配： ♀CCoo 白色 × ♂ccOO 白色 ↓ 子一代有色子一代用双隐性个体ccoo 测交。做了很多这样的交配，得到的后代中，有色68只，白色204只。问o —c 之间有连锁吗？如有连锁，交换值是多少？解：根据题意，上述交配： ♀ CCoo 白色 ccOO 白色 ♂

↓ 有色CcOo ccoo 白色 ↓ 有色C_O_ 白色（O_cc ，ooC_，ccoo ） 416820468=+ 4 3 68204204=+ 此为自由组合时双杂合个体之测交分离比。可见，c —o 间无连锁。 (若有连锁，交换值应为50％，即被测交之F1形成Co ：cO ：CO ：co ＝1 ：1 ：1 ：1的配子；如果这样，那么c 与o 在连锁图上相距很远，一般依该二基因是不能直接测出重组图距来的)。 4、双杂合体产生的配子比例可以用测交来估算。现有一交配如下：问：（1）独立分配时，P=？（2）完全连锁时，P=？（3）有一定程度连锁时，p=？解：题目有误，改为：)2 1( )21 (aabb aaBb Aabb AaBb p p p p -- （1）独立分配时，P = 1/4；（2）完全连锁时，P = 0；（3）有一定程度连锁时，p = r /2，其中r 为重组值。 5、在家鸡中，px 和al 是引起阵发性痉挛和白化的伴性隐性基因。今有一双因子杂种公鸡 al Px Al px 与正常母鸡交配，孵出74只小鸡，其中16只是白化。假定小鸡没有一只早期死亡，而px 与al 之间的交换值是10％，那么在小鸡4周龄时，显出阵发性痉挛时，（1）在白化小鸡中有多少数目显出这种症状，（2）在非白化小鸡中有多少数目显出这种症状？解：上述交配子代小鸡预期频率图示如下： ♀W Al Px al Px Al px ♂

遗传学(刘祖洞)下册部分章节答案

遗传学（刘祖洞）下册部分章节答案第九章遗传物质的改变（一）染色体畸变 1．什么是染色体畸变？答：染色体数目或结构的改变，这些改变是较明显的染色体改变，一般可在显微镜下看到，称为染色体变异或畸变。 2．解释下列名词：缺失；重复；倒位；易位答：缺失（deletion 或deficiency）——染色体失去了片段。重复（duplication 或repeat）——染色体增加了片段。倒位（inversion）——染色体片段作1800的颠倒，造成染色体内的重新排列。易位（translocation）——非同源染色体间相互交换染色体片段，造成染色体间的重新排列。 3．什么是平衡致死品系，在遗传学研究中，它有什么用处？答：同源染色体的两个成员各带有一个座位不同的隐性致死基因，由于两个致死基因之间不发生交换，使致死基因永远以杂合态保存下来，不发生分离的品系，叫平衡致死品系（balanced lethsl system）。在遗传研究过程中，平衡致死系可用于保存致死基因。 4．解释下列名词：（１）单倍体，二倍体，多倍体；（２）单体，缺体，三体；（３）同源多倍体，异源多倍体答：（1）凡是细胞核中含有一个完整染色体组的叫做单倍体（haploid）；含有两个染色体组的叫做二倍体（diploid）；超过两个染色体组的统称多倍体（polyploid）。（2）细胞核内的染色体数不是完整的倍数，通常以一个二倍体（2n）染色体数作为标准，在这个基础上增减个别几个染色体，称非整倍性改变。例如：2n-1是单体（monsomic），2n-2是缺体（nullisomic），2n+1是三体（trisomic）。（3）同源多倍体（autopolyploid）——增加的染色体组来自同一个物种的多倍体。异源多倍体（allopolyloid）——加倍的染色体组来自不同物种的多倍体，是两个不相同的种杂交，它们的杂种再经过染色体加倍而形成的。 5．用图解说明无籽西瓜制种原理。答：亲本西瓜（2n=22） ↓秋水仙素 4n ♀× 2n ♂ 嫩绿色，无条斑，如马铃瓜↓具有深绿色平行条斑，如解放瓜 4n母本上结了西瓜，瓢中长着3n种子，把3n种子种下，所结的无籽西瓜是无籽的，其果皮有深绿色平行条斑

遗传学课后习题及答案刘祖洞

第二章孟德尔定律 1、为什么分离现象比显、隐性现象有更重要的意义? 答:因为1、分离规律是生物界普遍存在的一种遗传现象,而显性现象的表现是相对的、有条件的;2、只有遗传因子的分离和重组，才能表现出性状的显隐性。可以说无分离现象的存在,也就无显性现象的发生。 2、在番茄中，红果色（Ｒ)对黄果色（r)是显性，问下列杂交可以产生哪些基因型，哪些表现型，它们的比例如何(1)RR×rr(２)Rr×rr (3）Rr×Rｒ（4）Rr×RR(５)rr×ｒr 3、下面是紫茉莉的几组杂交,基因型和表型已写明。问它们产生哪些配子?杂种后代的基因型和表型怎样?(1)Ｒｒ× RR （２）rr × Rr(３)Rr×Rｒ粉红红色白色粉红粉红粉红 4、在南瓜中，果实的白色(W)对黄色(w)是显性,果实盘状(D）对球状（d）是显性，这两对基因是自由组合的。问下列杂交可以产生哪些基因型，哪些表型,它们的比例如何？(1)WWＤD×wｗdd （2）XｗDd×wwdd(3)Wwｄd×wwＤd (4)Ｗｗｄd×WwDd

2／8ＷwDd,2／8Ｗｗdd, 1/8wwＤd，1/8wwdd ３／8白色、盘状,3/8白色、球状，1/８黄色、盘状，1/8黄色、球状５.在豌豆中，蔓茎(T)对矮茎(ｔ)是显性，绿豆荚（Ｇ）对黄豆荚(g)是显性,圆种子（R）对皱种子（r)是显性。现在有下列两种杂交组合,问它们后代的表型如何?(１)TTＧgＲｒ×ttGgrr（2）ＴtGgrr×ttGgｒr 解:杂交组合TTGgＲr×ｔtGgrr: 即蔓茎绿豆荚圆种子3/８，蔓茎绿豆荚皱种子３/８，蔓茎黄豆荚圆种子１／8,蔓茎黄豆荚皱种子1／8。杂交组合TtGgｒr×ttGgｒr: 即蔓茎绿豆荚皱种子３/8，蔓茎黄豆荚皱种子1／８,矮茎绿豆荚皱种子3/8，矮茎黄豆荚皱种子1/８。 6.在番茄中,缺刻叶和马铃薯叶是一对相对性状，显性基因C控制缺刻叶，基因型ｃｃ是马铃薯叶.紫茎和绿茎是另一对相对性状，显性基因A控制紫茎，基因型aa的植株是绿茎。把紫茎、马铃薯叶的纯合植株与绿茎、缺刻叶的纯合植株杂交，在F2中得到９∶3∶3∶1的分离比.如果把F1:（1)与紫茎、马铃薯叶亲本回交;(2)与绿茎、缺刻叶亲本回交;以及（3)用双隐性植株测交时，下代表型比例各如何?

遗传学第三版刘祖洞第二章练习题

名词解释5个，选择题10个，解答题5个，问答题2个一.名词解释 1.细胞：细胞是生物体形态结构和生命活动的基本单位，也是生长发育和遗传的基本单位。 2.细胞生物：以细胞为基本单位的生物；根据细胞核和遗传物质的存在方式不同又可以分为真核细胞和原核细胞。 3. 染色质：染色质是在间期细胞核内有由DNA、组蛋白、非组蛋白和少量RNA组成的，易被碱性染料着色的一种无定形物质 4. 着丝粒：着丝粒是细胞分裂时纺锤丝附着(attachment)的区域，又称为着丝点。 5. 姊妹染色单体：一条染色体的两个染色单体互称为姊妹染色单体。 6. 次缢痕：某些染色体的一个或两个臂上往往还具有另一个染色较淡的缢缩部位，称为次缢痕，通常在染色体短臂上。 7. 同源染色体：体细胞中形态结构相同、遗传功能相似的一对染色体称为同源染色体二.选择题 1.在洋葱根尖分生区有丝分裂过程中，染色体数目加倍和DNA数加倍分别发生在（ C ） A.间期、间期 B.间期、后期 C.后期、间期 D.前期、间期 2.一个动物细胞周期中，DNA数加倍、染色体数加倍、中心粒复制的时期依次为（ C ）① 间期②前期③中期④后期⑤末期 A.①②④ B.①③⑤ C.①④① D.②④⑤ 3. 保证两个子细胞中染色体形态和数目与母细胞完全相同的机制是（ B ） A.染色体的复制 B.着丝点的分裂 C.纺锤丝的牵引 D.以上三项均起作用 4. 在一个细胞周期中，DNA复制过程中的解旋发生在( A ) A．两条DNA母链之间 B．DNA子链与其互补的母链之间 C．两条DNA子链之间 D．DNA子链与其非互补母链之间 5. 下列有关细胞生命活动的叙述，正确的是（ B ） A．分裂期的细胞不进行DNA复制和蛋白质合成 B．免疫系统中的记忆细胞既有分化潜能又有自我更新能力 C．凋亡细胞内的基因表达都下降，酶活性减弱 D．原癌基因突变促使细胞癌变，抑癌基因突变抑制细胞癌变 6. 1.人类l号染色体长臂分为4个区，靠近着丝粒的为( B )。 A．O区B．1区 C．2区D．3区 7. 真核细胞中的RNA来源于( D )。 A．DNA复制B．DNA裂解 C．DNA转化D．DNA转录 E .DNA翻译 8. 染色体不分离( D ) A只是指姊妹染色单体不分离 B．只是指同源染色体不分离

刘祖洞遗传学第三版答案-第9章-数量性状遗传

出简单的比例，因此只能用统计方法分析。（3）有可能出现超亲遗传。数量性状遗传和质量性状遗传的主要区别：（1）数量性状是表现连续变异的性状，而质量性状是表现不连续变异的性状；（2）数量性状的遗传方式要比质量性状的遗传方式复杂的多，它是由许多基因控制的，而且它们的表现容易受环境条件变化的影响。 2.什么叫遗传率？广义遗传率？狭义遗传率？平均显性程度？解析：根据定义回答就可以了。参考答案：遗传率指亲代传递其遗传特性的能力，是用来测量一个群体内某一性状由遗传因素引起的变异在表现型变异中所占的百分率，即：遗传方差/总方差的比值。广义遗传率是指表型方差（Vp）中遗传方差（Ve）所占的比率。狭义遗传率是指表型方差（Vp）中加性方差（V A） V V〔在数量 / D A 性状的遗传分析中，对于单位点模型，可以用显性效应和加性效应的比值d/a来表示显性程度。但是推广到多基因系统时，∑d/∑a并不能说明任

刘祖洞-遗传学-第二版-课后答案

第二章孟德尔定律 1、为什么分离现象比显、隐性现象有更重要的意义？答：这是因为：（1）性状的分离规律是生物界普遍存在的一种遗传现象，而显性现象的表现是相对的、有条件的；（2）只有基因发生分离和重组，才能表现出性状的显隐性。可以说无分离现象的存在，也就无显性现象的发生。 2、在番茄中，红果色（R ）对黄果色（r ）是显性，问下列杂交可以产生哪些基因型，哪些表现型，它们的比例如何？（1）RR×rr （2）Rr×rr （3）Rr×Rr （4） Rr×RR （5）rr×rr 解： 3、下面是紫茉莉的几组杂交，基因型和表型已写明。问它们产生哪些配子？杂种后代的基因型和表型怎样？（1）Rr × RR （2）rr × Rr （3）Rr × Rr 粉红红色白色粉红粉红粉红解： 4、在南瓜中，果实的白色（W ）对黄色（w ）是显性，果实盘状（D ）对球状（d ）是显性，这两对基因是自由组合的。问下列杂交可以产生哪些基因型，哪些表型，它们的比例如何？（1）WWDD×wwdd （2）XwDd×wwdd （3）Wwdd×wwDd （4）Wwdd×WwDd 解： 5.在豌豆中，蔓茎（T ）对矮茎（t ）是显性，绿豆荚（G ）对黄豆荚（g ）是显性，圆种子（R ）对皱种子（r ）是显性。现在有下列两种杂交组合，问它们后代的表型如何？（1）TTGgRr×ttGgrr （2）TtGgrr×ttGgrr 解：杂交组合TTGgRr × ttGgrr ：

即蔓茎绿豆荚圆种子3/8，蔓茎绿豆荚皱种子3/8，蔓茎黄豆荚圆种子1/8，蔓茎黄豆荚皱种子1/8。杂交组合TtGgrr × ttGgrr：即蔓茎绿豆荚皱种子3/8，蔓茎黄豆荚皱种子1/8，矮茎绿豆荚皱种子3/8，矮茎黄豆荚皱种子1/8。 6.在番茄中，缺刻叶和马铃薯叶是一对相对性状，显性基因C控制缺刻叶，基因型cc是马铃薯叶。紫茎和绿茎是另一对相对性状，显性基因A控制紫茎，基因型aa的植株是绿茎。把紫茎、马铃薯叶的纯合植株与绿茎、缺刻叶的纯合植株杂交，在F2中得到9∶3∶3∶1的分离比。如果把F1：（1）与紫茎、马铃薯叶亲本回交；（2）与绿茎、缺刻叶亲本回交；以及（3）用双隐性植株测交时，下代表型比例各如何？解：题中F2分离比提示：番茄叶形和茎色为孟德尔式遗传。所以对三种交配可作如下分析： (1) 紫茎马铃暮叶对F1的回交： (2) 绿茎缺刻叶对F1的回交：（3）双隐性植株对F l测交： AaCc × aacc

刘祖洞遗传学第三版答案_第10章_染色体畸变

第十章遗传物质的改变（1）- 染色体畸变 1 什么叫染色体畸变？解答：染色体畸变是指染色体发生数目或结构上的改变。（1 ）染色体结构畸变指染色体发生断裂，并以异常的组合方式重新连接。其畸变类型有缺失、重复、倒位、易位。（ 2 ）染色体数目畸变指以二倍体为标准所出现的成倍性增减或某一对染色体数目的改变统称为染色体畸变。前一类变化产生多倍体，后一类称为非整倍体畸变。 2 解释下列名词：（1）缺失；（2）重复；（3）倒位；（4）易位。解答：缺失：缺失指的是染色体丢失了某一个区段。重复：重复是指染色体多了自己的某一区段倒位：倒位是指染色体某区段的正常直线顺序颠倒了。易位：易位是指某染色体的一个区段移接在非同源的另一个染色体上。 3 什么叫平衡致死品系？在遗传学研究中，它有什么用处？解答：紧密连锁或中间具有倒位片段的相邻基因由于生殖细胞的同源染色体不能交换，所以可以产生非等位基因的双杂合子，这种利用倒位对交换抑制的效应，保存非等位基因的纯合隐性致死基因，该品系被称为平衡致死系。平衡致死的个体真实遗传，并且它们的遗传行为和表型表现模拟了具有纯合基因型的个体，因此平衡致死系又称永久杂种。平衡致死品系在遗传学研究中的用处： 1）利用所谓的交换抑制子保存致死突变品系- 平衡致死系可以检测隐形突变（2）用于实验室中致死、半致死或不育突变体培养的保存（ 3 ）检测性别 4解释下列名词：（1）单倍体，二倍体，多倍体。（2）单体，缺体，三体。

（3）同源多倍体，异源多倍体。解答：（1）单倍体（haploid）:是指具有配子体染色体数目的个体。二倍体（diploid）:细胞核内具有两个染色体组的生物为二倍体。多倍体（polyploid）:细胞中有3个或3个以上染色体组的个体称为多倍体。（2）单体（monosomic）:是指体细胞中某对染色体缺少一条的个体（2n -1 ）缺体（nu llosomic）:是指生物体细胞中缺少一对同源染色体的个体（2n —2），它仅存在于多倍体生物中，二倍体生物中的缺体不能存活。三体（trisomic）:是指体细胞中的染色体较正常2n个体增加一条的变异类型，即某一对染色体有三条染色体（2n + 1 ）。（3）同源多倍体：由同一染色体组加倍而成的含有三个以上的染色体组的个体称为同源多倍体。异源多倍体：是指体细胞中具有2个或2个以上不同类型的染色体组。 5用图解说明无籽西瓜制种原理。解答：优良二倍体西瓜品种 1，人工加倍早四倍体X二倍体$ 早三倍体X二倍体$ 样联会紊乱三倍体无籽西瓜 6异源八倍体小黑麦是如何育成的？解答：普通小麦X黑麦

刘祖洞遗传学第三版答案第13章细胞质和遗传

第十三章细胞质和遗传 1．母性影响和细胞质遗传有什么不同？答： 1）母性影响是亲代核基因的某些产物或者某种因子积累在卵细胞的细胞质中，对子代某些性状的表现产生影响的现象。这种效应只能影响子代的性状，不能遗传。因此F1代表型受母亲的基因型控制，属于细胞核遗传体系；细胞质遗传是细胞质中的DNA或基因对遗传性状的决定作用。由于精卵结合时，精子的细胞质往往不进入受精卵中，因此，细胞质遗传性状只能通过母体或卵细胞传递给子代，子代总是表现为母本性状，属于细胞质遗传体系，2）母性影响符合孟德尔遗传规律；细胞质遗传是非孟德尔式遗传。 3）母性遗传杂交后代有一定的分离比, 只不过是要推迟一个世代而已；细胞质遗传杂交后代一般不出现一定的分离比。 2．细胞质基因和核基因有什么相同的地方，有什么不同的地方？答： 1）相同：细胞核遗传和细胞质遗传各自都有相对的独立性。这是因为，尽管在细胞质中找不到染色体一样的结构，但质基因与核基因一样，可以自我复制，可以控制蛋白质的合成，也就是说，都具有稳定性、连续性、变异性和独立性。 2）不同： A. 细胞质和细胞核的遗传物质都是DNA分子，但是其分布的位置不同。细胞核遗传的遗传物质在细胞核中的染色体上；细胞质中的遗传物质在细胞质中的线粒体和叶绿体中。 B. 细胞质和细胞核的遗传都是通过配子，但是细胞核遗传雌雄配子的核遗传物质相等，而细胞质遗传物质主要存在于卵细胞中； C. 细胞核和细胞质的性状表达都是通过体细胞进行的。核遗传物质的载体（染色体）有均分机制，遵循三大遗传定律；细胞质遗传物质（具有DNA的细胞器如线粒体、叶绿体等）没有均分机制，是随机分配的。 D. 细胞核遗传时，正反交相同，即子一代均表现显性亲本的性状；细胞质遗传时，正反交不同，子一代性状均与母本相同，即母系遗传。 3．在玉米中，利用细胞质雄性不育和育性恢复基因，制造双交种，有一个方式是这样的：先把雄性不育自交系A【（S）rfrf】与雄性可育自交系B【（N）rfrf】杂交，得单交种AB，把雄性不育自交系C【（S）rfrf】与雄性可育自交系D【（N）RfRf】杂交，得单交种CD。然后再把两个单交种杂交，得双交种ABCD，问双交种的基因型和表型有哪几种，它们的比例怎样？解： A【（S）rfrf】? B【（N）rfrf】C【（S）rfrf】? D【（N）RfRf】 ↓↓ AB【（S）rfrf】?CD【（S）Rfrf】 ↓ 基因型：1/2【(S)rfrf】1/2【(S)Rfrf】表型：雄性不育雄性可育 4．“遗传上分离的”小菌落酵母菌在表型上跟我们讲过的“细胞质”小菌落酵母菌相似。当一个遗传上分离的小菌落酵母菌与一个正常酵母菌杂交，二倍体细胞是正常的，以后形成子囊孢子时，每个子囊中两个孢子是正常的，两个孢子产生小菌落酵母菌。用

刘祖洞的遗传学(第二版)笔记

遗传学总复习第一章绪论遗传学及分支学科遗传学的发展、任务第二章孟德尔定律 key words：反应规范（reaction norm）、等位基因（allele）、复等位基因（multiple alleles）、表型模写（phenocopy）、外现率（penetrance）、互补基因（complementary gene）、抑制基因（suppress gene）、表现度（expressivity）、抑制基因（inhibitor）、上位效应（epistatic effect ）、叠加效应(duplicate effect) 一、积加概率卡平方测验三大定律系谱符号概率的应用二、遗传的染色体学说三、细胞分裂中染色体的变化核型染色体形态分析四、基因的作用及与环境的关系五、基因与环境六、一因多效、多因一效七、显、隐性的相对性八、致死基因九、ABO血型、Rh血型、HLA血型、血型不亲和、孟买型与类孟买型十、非等位基因间的作用：互补、抑制、显性上位、隐性上位第三章连锁遗传分析与染色体作图 key word：伴性遗传（sex-linked inheritance）、从性遗传（sex-condition inheritance）、限雄遗传（holandric inheritance）、基因组印迹（genomic imprinting）、 Lyon 假说、动态突变（dynamic mutation）、拟常染色体基因（pseudoautosomal gene）、性分化（sex differentiation）、脆性X综合症（fragile X syndrome ）、睾丸决定因子（testis-determining factor ）一、性别决定与伴性遗传

刘祖洞遗传学第三版答案第10章染色体畸变

第十章遗传物质的改变（1）-染色体畸变 1 什么叫染色体畸变？解答：染色体畸变是指染色体发生数目或结构上的改变。（1）染色体结构畸变指染色体发生断裂，并以异常的组合方式重新连接。其畸变类型有缺失、重复、倒位、易位。（2）染色体数目畸变指以二倍体为标准所出现的成倍性增减或某一对染色体数目的改变统称为染色体畸变。前一类变化产生多倍体，后一类称为非整倍体畸变。 2 解释下列名词：（1）缺失；（2）重复；（3）倒位；（4）易位。解答：缺失：缺失指的是染色体丢失了某一个区段。重复：重复是指染色体多了自己的某一区段倒位：倒位是指染色体某区段的正常直线顺序颠倒了。易位：易位是指某染色体的一个区段移接在非同源的另一个染色体上。 3 什么叫平衡致死品系？在遗传学研究中，它有什么用处？解答：紧密连锁或中间具有倒位片段的相邻基因由于生殖细胞的同源染色体不能交换，所以可以产生非等位基因的双杂合子，这种利用倒位对交换抑制的效应，保存非等位基因的纯合隐性致死基因，该品系被称为平衡致死系。平衡致死的个体真实遗传，并且它们的遗传行为和表型表现模拟了具有纯合基因型的个体，因此平衡致死系又称永久杂种。平衡致死品系在遗传学研究中的用处：（1）利用所谓的交换抑制子保存致死突变品系-平衡致死系可以检测隐形突变（2）用于实验室中致死、半致死或不育突变体培养的保存（3）检测性别 4 解释下列名词：（1）单倍体，二倍体，多倍体。（2）单体，缺体，三体。（3）同源多倍体，异源多倍体。解答：（1）单倍体(haploid)：是指具有配子体染色体数目的个体。

二倍体(diploid)：细胞核内具有两个染色体组的生物为二倍体。多倍体(polyploid)：细胞中有3个或3个以上染色体组的个体称为多倍体。（2）单体(monosomic)：是指体细胞中某对染色体缺少一条的个体（2n－1）。缺体(nullosomic)：是指生物体细胞中缺少一对同源染色体的个体（2n －2），它仅存在于多倍体生物中，二倍体生物中的缺体不能存活。三体(trisomic)：是指体细胞中的染色体较正常2n个体增加一条的变异类型，即某一对染色体有三条染色体（2n＋1）。（3）同源多倍体：由同一染色体组加倍而成的含有三个以上的染色体组的个体称为同源多倍体。异源多倍体：是指体细胞中具有2个或2个以上不同类型的染色体组。 5 用图解说明无籽西瓜制种原理。解答：优良二倍体西瓜品种人工加倍 ♀三倍体×二倍体♂ 联会紊乱三倍体无籽西瓜 6 异源八倍体小黑麦是如何育成的？解答：普通小麦×黑麦 (42) AABBDD RR(14) ABDR 染色体加倍 AABBDDRR(56) 异源八倍体小黑麦 7 何以单倍体的个体多不育？有否例外？举例。解答：单倍体个体多是不育的，因为单倍体在减数分裂时,由于染色体成单价体存在,没有相互联会的同源染色体,所以最后将无规律地分离到配子中去,结果极大多数不能发育成有效配子,因而表现高度不育。有时也存在特殊的情况，例如四

遗传学(下)刘祖洞第二版

第九章遗传物质的改变（一）染色体畸变应用前几章中讲过的一些遗传学基本定律，如分离和组合、连锁与交换，可在子代中得到亲代所不表现的新性状，或性状的新组合。但这些“新”性状，追溯起来并不是真正的新性状，都是它们祖先中原来有的。只有遗传物质的改变，才出现新的基因，形成新的基因型，产生新的表型。遗传物质的改变，称作突变（mutation）。突变可以分为两大类：（1）染色体数目的改变和结构的改变，这些改变一般可在显微镜下看到；（2）基因突变或点突变（genic or pointmutations），这些突变通常在表型上有所表达。但在传统上，突变这一术语留给基因突变，而较明显的染色体改变，称为染色体变异或畸变（chromosomal variations or aberrations）。第一节染色体结构的改变因为一个染色体上排列着很多基因，所以不仅染色体数目的变异可以引起遗传信息的改变，而且染色体结构的变化，也可引起遗传信息的改变。一般认为，染色体的结构变异起因于染色体或它的亚单位——染色单体的断裂（breakage）。每一断裂产生两个断裂端，这些断裂端可以沿着下面三条途径中的一条发展：（1）它们保持原状，不愈合，没有着丝粒的染色体片段（seg-ment）最后丢失。（2）同一断裂的两个断裂端重新愈合或重建（restitution），回复到原来的染色体结构。（3）某一断裂的一个或两个断裂端，可以跟另一断裂所产生的断裂端连接，引起非重建性愈合（nonrestitution union）。依据断裂的数目和位置，断裂端是否连接，以及连接的方式，可以产生各种染色体变异，主要的有下列四种（图9－1）：（1）缺失（deletion或deficiency）——染色体失去了片段；

刘祖洞(遗传学)课后习题标准答案!全面版

刘祖洞《遗传学》参考答案全面版 ?第二章孟德尔定律１、为什么分离现象比显、隐性现象有更重要的意义? (1）分离规律是生物界普遍存在的一种遗传现象，而显性现象的表现是相对的、有条件的;? (2）答:因为? 只有遗传因子的分离和重组，才能表现出性状的显隐性。可以说无分离现象的存在,也就无显性现象的发生。?2、解:序号杂交基因型表现型 (1）RR×ｒr Rr 红果色?(2) Ｒr×ｒr 1/2Rｒ，１／２rr 1／2红果色,１/2黄果色 (3)Rr×Rr 1/4RR,2/4Rr，1/４rｒ３/4红果色,1/4黄果色 (4)Rｒ×RR 1／２RR，1／2Rr 红果色 (5）ｒr×rr rｒ黄果色?3、下面是紫茉莉的几组杂交，基因型和表型已写明。问它们产生哪些配子?杂种后代的基因型和表型怎样？（１)Ｒr× RＲ（2)rr ×Rｒ（3)Rr ×Ｒr 粉红红色白色粉红粉红粉红?解:序号杂交配子类型基因型表现型（1）Rr× RR Ｒ,r;R 1/２ＲＲ,1/2Rr 1/２红色,1/2粉红（2）rr × Rｒr；R,r1/2Rｒ,１/2rr 1/2粉红，1／2白色 4、在南瓜中，果实的白色(W)（3) Rr× Rr R，r1/4RR,２/4Rr,１/4rｒ1/4红色,２/4粉色,1/4白色? 对黄色(w)是显性,果实盘状(D)对球状（ｄ)是显性,这两对基因是自由组合的。问下列杂交可以产生哪些基因型，哪些表型,它们的比例如何？ (1)WWDD×ｗwdｄ(2）ＸwＤd×wwdd （3）Ｗwｄｄ×wwDd(4)Wwｄd×WwDd 解:?序号杂交基因型表现型１WWDD×wwｄd WwDd 白色、盘状果实 2WwＤd×wwdd １/４WwDd，1/4Ｗwdd,1/4wwDd，１/4wwdd,1/4白色、盘状，1/4白色、球状,1/ 2ｗｗDd×wwｄd 1/２wwＤｄ,１/2wwdd 1/2黄色、盘状，1／２黄色、４黄色、盘状，１/4黄色、球状? 球状 3 Wwｄd×wwDd １/4WｗDｄ,1/４Wwdd，1／４wwDd,１/４ｗｗdd，１/4白色、盘状,1/4白色、球 4Wwｄd×WwＤｄ１/8WWDd，1／8WWdd，2/8WwDd,２／状,1/4黄色、盘状，１／4黄色、球状? 8Wwdd，１/8wwDd,1／8wｗｄd 3/８白色、盘状，3/8白色、球状，１/8黄色、盘状,1/8黄色、球状 ?５.在豌豆中，蔓茎(T）对矮茎（t)是显性,绿豆荚(G)对黄豆荚（g）是显性，圆种子（R）对皱种子(r）是显性。现在有下列两种杂交组合,问它们后代的表型如何? (1）TＴGgRr×ttGgrr (２)TtＧgrr×ｔtＧgrｒ解:杂交组合TTＧgRr × ttGgｒr：即蔓茎绿豆荚圆种子3/8,蔓茎绿豆荚皱种子3/８,蔓茎黄豆荚圆种子１/8,蔓茎黄豆荚皱种子1/8。杂交组合ＴtGgｒr ×ttGｇrｒ：即蔓茎绿豆荚皱种子3/８,蔓茎黄豆荚皱种子1/8，矮茎绿豆荚皱种子3／8,矮茎黄豆荚皱种子１/8。6．在番茄中,缺刻叶和马铃薯叶是一对相对性状,显性基因C控制缺刻叶，基因型cｃ是马铃薯叶。紫茎和绿茎是另一对相对性状,显性基因A控制紫茎,基因型aa的植株是绿茎。把紫茎、马铃薯叶的纯合植株与绿茎、缺刻叶的纯合植株杂交，在F2中得到9∶３∶3∶１的分离比。如果把F１：(１)与紫茎、马铃薯叶亲本回交;(2）与绿茎、缺刻叶亲本回交；以及(３)用双隐性植株测交时,下代表型比例各如何？解:题中F2分离比提示：番茄叶形和茎色为孟德尔式遗传。所以对三种交配可作如下分析：?（1) 紫茎马铃暮叶对F1的回交：（2)绿茎缺刻叶对F1的回交：（3)双隐性植株对Ｆl测交：

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