一含有两个开放阅读框架的cDNA片段的发现
作者：姜颖 于永利g$si, 百拇医药
单位：g$si, 百拇医药
关键词：基因；重叠；大衣壳蛋白；人疱疹病毒3；外膜蛋白rck；沙门氏菌g$si, 百拇医药
免疫学杂志000501g$si, 百拇医药
[摘 要] 目的 分离人知的cDNAs。方法 采用RT-PCR自人的肝脏细胞分离到一约1，200bp的cDNA片段，将其克隆测序。结果 在这一cDNA片段中包含了两个开放阅读框架。结论 一个开放阅读框架代表与人疱疹病毒大衣壳蛋白同源的多肽，另一个代表与沙门氏菌外膜蛋白rck同源的多肽。g$si, 百拇医药
[中图分类号] R342 [文献标识码] Ag$si, 百拇医药
A cDNA fragment containing two open reading framesg$si, 百拇医药
JIANG Ying， YU Yong-lig$si, 百拇医药
(Department of immunology，Norman Bethune University of Medical Sciences，Changchun 130021，China)g$si, 百拇医药
[Abstract] Object To isolate human unkown cDNAs.Methods With RT-PCR primed by specific human angiostatin primers，a cDNA fragment about 1 200 bp was isolated，cloned and partly sequenced. Results Two open reading frames (ORFs) in the fragment have been found.Conclusion One putative peptide deduced from the ORFs is homologous to large tegument protein of human herpes virus 3 and the other homologous to outer membrene protein rck of salmoella typhimurium.
[Key words] gene；overlapping；large tegument protein；human herpesvirus 3；outer membrane protein rck；salmonella typhimuriumpa., 百拇医药
[Article ID] 1000-8861(2000)05-0321-04pa., 百拇医药
Angiostatin is a potent angiogenesis inhibitor which has been identified as an internal fragment of plasminogen.Since tumor growth is exclusively dependent on angiogenesis，angiostatin can be used as a promising tumor inhibitor.We tried to isolate human angiostatin cDNA utilizing RT-PCR with a pair of specific primers from total cDNA of human liver.A cDNA fragment about 1 200 bp was amplified，cloned，partly sequenced and analyzed.Two open reading frames can be found within the cDNA.pa., 百拇医药
1 Materials and Methodspa., 百拇医药
1.1 Materials Taq DNA polymerase，AMV reverse transcriptase，Rnase inhibitor，T4 DNA ligase，restriction endonuclease EcoRⅠ were from obtained Promega Co.TRIzol Reagent from GIBCOBRL Co.Geneclean kit from Department of Molecular Biology，Norman Bethune University of Medical Sciences.Plasmid pGEM-T easy vector and JM109 from Promega Co.PCR primers were synthesized by Shanghai Sangon Co.E.coli strain.Human liver samples were provided by the Third Clinical Hospital of Norman Bethune University of Medical Sciences with patients’ permission.
1.2 Primers design The primers were designed based on the published cDNA sequence of human plasminogen in Genbank.The primers(5’cgcg-tcgggaattcttatttgaaaagaaag.3’atatgtcgacttaaagcaggacaacaggc)contain ECoRⅠ and SalⅠ restriction sites respectively.n, http://www.100md.com
1.3 Isolation of total RNA from human liver cells Total cytoplasmic RNA was isolated from human embryonic liver cells by TRIzol Reagent following the protocols provided with the reagent.The RNA precipitate was dissolved in 9 μl DEPC H2O.n, http://www.100md.com
1.4 Synthesis of first strand cDNA and PCR amplification Add oligo-dT(0.05 g/ml)1 μl to the resultant RNA.Incubate at 65 °C for 10 min and then in ice-cold bath for 2 min.Add the following reagents to the reaction：5×AMV reverse trans-criptase buffer 4.7 μl，Rnasin(40 u/ml)0.5 μl，dNTP(5 mmol/L)8 μl，AMV(200 u/l)0.5 μl.Incubate at 37 °C for 1 hour.2 μl of cDNA were used per PCR reaction.PCR protocol：94 °C，1 min；55 °C，1 min；72 A 90 s，35 cycles，final extention，72 °C 10 min.10 μl of the amplified products were visualized by 1 % agarose gel electrophoresis.n, http://www.100md.com
1.5 Cloning and partly sequencing of the PCR product 10 μl PCR product was purified from agarose gel with Geneclean Kit.Clone the fragment into TA cloning vector pGEM-T easy.Transform the ligated product into JM109 competent cells.The recombinant colonies were selected by blue-white screening on IPTG/X-gal plates.The plasmid was extracted from positive colonies and digested with EcoRⅠ to confirm recombination.The insert in recombinant plasmid was partly sequenced by Dalian TaKaPa Corporation.
1.6 Analysis of the fragment The sequenced fragment was analyzed with BLASTX，BLASTN in Genbank and MultiIdent，ProtPram，PSORT，SignalP，ProtScale，PredictProt etc.in ExPASy through Internet.9, 百拇医药
2 Results9, 百拇医药
2.1 Isolation，cloning and sequencing of the PCR product A cDNA fragment about 1 200 bp was amplified with RT-PCR，cloned into pGEM-T easy vector(see Figure 1)and partly sequenced(see Figure 2).
9, 百拇医药
Fig 1Isolation and cloning of the cDNA fragment9, 百拇医药
Lane 1.DNA marker：2 000，1 000，750，500，250，1009, 百拇医药
Lane 2.PCR product of angiostatin9, 百拇医药
Lane 3.released insert from recycled TA cloning vector
9, 百拇医药
Fig 2The sequence of the cDNA9, 百拇医药
2.2 Analysis of the cDNA The cDNA fragment is not homologous to angiostatin except primer site.There were two incomplete open reading frames within the sequence.The sequences of the encoding peptides were deduced.The correspon-ding peptides are designated peptide 1 and peptide 2 individually.Searching with BLAST and BLASTN in Genbank shows that there are no high homology in terms of nucleotides，but they do have homology in terms of amino acids.The two putative peptides are 21 percent-homologous to large tegument protein of the human herpesvirus 3 and 12 percent-homologous to outer membrane protein rck of Salmonella typhimurium respectively(see figure 3，4)，and are analyzed in Swissprot.
Fig 3Sequence and similarities of peptide 1
g@y, http://www.100md.com
Fig 4Sequence and similarities of peptide 2g@y, http://www.100md.com
Peptide 1 is about 100 a.a long.It’s molecular weight is 10.823 kD and theoretical PI is 8.88.Since the stop codon in the ORF is within the PCR primer，so we are less sure whether the stop codon is a real or an artificial one.So peptide 1 we deduced from ORF may only be a part of an unidentified protein.It is predicted as a transmembrane protein with the probability of 44.4% and contains a transmembrane helix(from 11 a.a to 29 a.a)with N-terminus inside.The predicted O-glycosylation sites are Thr(No.47 and 65) and Ser(No.51).Peptide 2 is 99 a.a long with molecular weight 11.037 kD and theoretical PI5.29.It’s considered to be a transmembrane protein as well.The transmembrane helix spans from No.2 a.a to 22 a.a with N-terminus inside.Thr(52)is the preferred O-glycosylation site.g@y, http://www.100md.com
3 Discussiong@y, http://www.100md.com
3.1 The cDNA fragment isolated seems ubiquitous in human embryonic liver The cDNA fragment seems ubiquitous in human embryonic liver tissue.It could be isolated from liver samples of three embryos while not from other human tissue samples，such as adult liver，prostate，heart as well as embryonic heart，kidney，lung and brain.It can be inferred that the cDNA fragment was a transi-tory product in the particular stage during the development of embryonic liver.This suggests the possible relation between the expression of the cDNA and the development of liver.
3.2 There are two open reading frames(ORFs)within the cDNA fragment The peptide 1 is homologous to large tegument protein of the human herpesvirus 3.As a transmembrane protein，the prediction of peptide 1 from Swissprot coordinates with large tegument protein.The appearance of large tegument protein is the evidence of viral infection.VAN LOON NM performed direct sequence analysis of polymerase chain reaction-amplified DNA fragments from the large tegument protein(LTP)gene of human herpesvirus 6(HHV-6)with use of uncultured peripheral blood mononuclear cells(PBMCs)from four mother/infant pairs.In two cases，LTP gene sequences were identical in paired mother/infant specimens，thus suggesting that mother-to-infant transmission of HHV-6 may have occurred.The genetic stability of HHV-6 strains was confirmed by the fact that there was no difference between amplified DNA fragments from sequential PBMC samples from two out of two infants analyzed.In contrast ，a change in the amplified viral strain was detected in an infant who had reinfection with HHV-6 variant B(HHV-6B).Furthermore，HHV-6B strains concurrently amplified from saliva and PBMCs from an adult were found to be different.The data suggest that HHV-6 may be frequently transmitted from mother-to-infant and that reinfection with HHV-6B may occur[1].
Part sequence of the peptide 2 is much similar to the outer membrane protein rck(identities 40%)of Salmonella typhimurium.The 17kDa rck is encoded on the Salmonella typhimurium virulence plasmid and confers serum resistance and euka-ryotic cell invasion.The complement resistance mediated by rck is associated with a failure to form fully polymerized tubular membrane attack complexes[2].+4, 百拇医药
The two peptides are both transmembrane proteins with amino termini inside，therefore they can be classified into type Ⅱ membrane proteins.As we know，most transmembrane proteins are receptors and/or signal transduction proteins which are being studied intensively in the world.So the peptides themselves are worthy of further investigation as transmembrane proteins.+4, 百拇医药
3.3 Why are human genes homo-logous to human herpesvirus 3 and Salmonella typhimurium？ The homologous genes between virus and human have been widely accepted as oncogenes.So far we haven’t found any report about existence of human herpesvirus 3 and Salmonella typhimurium homo-logous sequences in the human genome.Why are human genes homologous to microorganisms genes？It is conceivable that genes between virus and animals are homologous because virus genes can integrate into animals’ chromosome by recombination or gain animals’ genes by retrotrans-cription，although whether the virus capture the genes from animals or the virus genes are captured by animals is uncertain.The existence of bacteria-homologous sequence in human genome is a thing hard to explain yet.Since all of the animals and microorganisms develop their genes from the same ancestor called “progenote”[3]，we may reach the explanation of the phenomena in view of evolution someday.The significance of observation reported in this article deserves further research.In addition，other mechanisms by which some genes“flow” between animals and microorganisms might exist as well and inspire us to explore.
3.4 The two ORFs are overlapped Another phenomenon needed mention here is that the two ORFs encoding the peptides homologous to LTP of human herpesvirus 3 and outer membrane protein rck of Salmonella typhimurium are in different reading frames and part of them are overlapped.The overlapping genes were considered to be specific to virus and phage.But recently they have been identified in mammals as well，such as human being[4～7]，rat[8]and mouse[9]etc.Our findings support the evidence that the overlapping genes also exist in human genome.5?, 百拇医药
[Biography] Jiang Ying(1973-)，female，born in Tonghua City， Jilin Province，Posdoc.Ph.D.，field of research：molecular immunology，Tel：(0431)5645911-6462；E-mail：nbumsjc＠public.cc.jl.cn5?, 百拇医药
姜颖(白求恩医科大学免疫学教研室，吉林 长春 130021)5?, 百拇医药
于永利(白求恩医科大学免疫学教研室，吉林 长春 130021)5?, 百拇医药
[REFERENCES]5?, 百拇医药
[1] VAN LOON NM，GUMMULURU S，SHERWOOD DJ，et al.Direct sequence analysis of human herpesvirus 6(HHV-6)sequences from infants and comparison of HHV-6 sequences from mother/infant pairs[J].Clin Infect Dis，1995，21：1 017-1 020.
[2] HEFFERNAN EJ，REED S，HACKETT J，et al.Mechanism of resistance to complement-mediated killing of bacteria encoded by the Salmonella typhimurium virulence plasmid gene rck[J].J Clin Invest，1992，90：953-957.$(rjs, http://www.100md.com
[3] 孙乃恩，孙东旭，朱德煦.分子遗传学[M].南京：南京大学出版社，1996.76-80.$(rjs, http://www.100md.com
[4] SHINTANI S，O’HUIGIN C，TOYOSAWA S，et al.Origin of gene overlap：the case of TCP1 and ACAT2[J].Genetics，1999，152：743-745.$(rjs, http://www.100md.com
[5] BACHMAN NJ，WU W，SCHMIDT TR，et al.The 5’ region of the COX4 gene contains a novel overlapping gene，NOC4[J].Mamm Genome，1999，10：506-512.$(rjs, http://www.100md.com
[6] COOPER PR，SMILINICH NJ，DAY CD，et al.Divergently transcribed overlapping genes expressed in liver and kidney and located in the 11p15.5 imprinted domain[J].Genomics，1998，49：38-51.$(rjs, http://www.100md.com
[7] HERZOG H，DARBY K，BALL H，et al.Overlapping gene structure of the human neuropeptide Y receptor subtypes Y1 and Y5 suggests coordinate transcriptional regulation[J].Genomics，1997，41：315-319.$(rjs, http://www.100md.com
[8] JOSEPH DR.The rat androgen-binding protein(ABP/SHBG)gene contains triplet repeats similar to unstable triplets：evidence that the ABP/SHBG and the fragile X-related 2 genes overlap[J].Steroids，1998，63：2-4.$(rjs, http://www.100md.com
[9] SMITH DP，SPICER J，SMITH A，et al. The mouse Peutz-Jeghers syndrome gene Lkbl encodes a nuclear protein kinase[J].Hum Mol Genet，1999，8：1 479-1 485.$(rjs, http://www.100md.com
[Received date] 1999-11-25； [Revised date] 2000-04-20(姜颖 于永利)
单位：g$si, 百拇医药
关键词：基因；重叠；大衣壳蛋白；人疱疹病毒3；外膜蛋白rck；沙门氏菌g$si, 百拇医药
免疫学杂志000501g$si, 百拇医药
[摘 要] 目的 分离人知的cDNAs。方法 采用RT-PCR自人的肝脏细胞分离到一约1，200bp的cDNA片段，将其克隆测序。结果 在这一cDNA片段中包含了两个开放阅读框架。结论 一个开放阅读框架代表与人疱疹病毒大衣壳蛋白同源的多肽，另一个代表与沙门氏菌外膜蛋白rck同源的多肽。g$si, 百拇医药
[中图分类号] R342 [文献标识码] Ag$si, 百拇医药
A cDNA fragment containing two open reading framesg$si, 百拇医药
JIANG Ying， YU Yong-lig$si, 百拇医药
(Department of immunology，Norman Bethune University of Medical Sciences，Changchun 130021，China)g$si, 百拇医药
[Abstract] Object To isolate human unkown cDNAs.Methods With RT-PCR primed by specific human angiostatin primers，a cDNA fragment about 1 200 bp was isolated，cloned and partly sequenced. Results Two open reading frames (ORFs) in the fragment have been found.Conclusion One putative peptide deduced from the ORFs is homologous to large tegument protein of human herpes virus 3 and the other homologous to outer membrene protein rck of salmoella typhimurium.
[Key words] gene；overlapping；large tegument protein；human herpesvirus 3；outer membrane protein rck；salmonella typhimuriumpa., 百拇医药
[Article ID] 1000-8861(2000)05-0321-04pa., 百拇医药
Angiostatin is a potent angiogenesis inhibitor which has been identified as an internal fragment of plasminogen.Since tumor growth is exclusively dependent on angiogenesis，angiostatin can be used as a promising tumor inhibitor.We tried to isolate human angiostatin cDNA utilizing RT-PCR with a pair of specific primers from total cDNA of human liver.A cDNA fragment about 1 200 bp was amplified，cloned，partly sequenced and analyzed.Two open reading frames can be found within the cDNA.pa., 百拇医药
1 Materials and Methodspa., 百拇医药
1.1 Materials Taq DNA polymerase，AMV reverse transcriptase，Rnase inhibitor，T4 DNA ligase，restriction endonuclease EcoRⅠ were from obtained Promega Co.TRIzol Reagent from GIBCOBRL Co.Geneclean kit from Department of Molecular Biology，Norman Bethune University of Medical Sciences.Plasmid pGEM-T easy vector and JM109 from Promega Co.PCR primers were synthesized by Shanghai Sangon Co.E.coli strain.Human liver samples were provided by the Third Clinical Hospital of Norman Bethune University of Medical Sciences with patients’ permission.
1.2 Primers design The primers were designed based on the published cDNA sequence of human plasminogen in Genbank.The primers(5’cgcg-tcgggaattcttatttgaaaagaaag.3’atatgtcgacttaaagcaggacaacaggc)contain ECoRⅠ and SalⅠ restriction sites respectively.n, http://www.100md.com
1.3 Isolation of total RNA from human liver cells Total cytoplasmic RNA was isolated from human embryonic liver cells by TRIzol Reagent following the protocols provided with the reagent.The RNA precipitate was dissolved in 9 μl DEPC H2O.n, http://www.100md.com
1.4 Synthesis of first strand cDNA and PCR amplification Add oligo-dT(0.05 g/ml)1 μl to the resultant RNA.Incubate at 65 °C for 10 min and then in ice-cold bath for 2 min.Add the following reagents to the reaction：5×AMV reverse trans-criptase buffer 4.7 μl，Rnasin(40 u/ml)0.5 μl，dNTP(5 mmol/L)8 μl，AMV(200 u/l)0.5 μl.Incubate at 37 °C for 1 hour.2 μl of cDNA were used per PCR reaction.PCR protocol：94 °C，1 min；55 °C，1 min；72 A 90 s，35 cycles，final extention，72 °C 10 min.10 μl of the amplified products were visualized by 1 % agarose gel electrophoresis.n, http://www.100md.com
1.5 Cloning and partly sequencing of the PCR product 10 μl PCR product was purified from agarose gel with Geneclean Kit.Clone the fragment into TA cloning vector pGEM-T easy.Transform the ligated product into JM109 competent cells.The recombinant colonies were selected by blue-white screening on IPTG/X-gal plates.The plasmid was extracted from positive colonies and digested with EcoRⅠ to confirm recombination.The insert in recombinant plasmid was partly sequenced by Dalian TaKaPa Corporation.
1.6 Analysis of the fragment The sequenced fragment was analyzed with BLASTX，BLASTN in Genbank and MultiIdent，ProtPram，PSORT，SignalP，ProtScale，PredictProt etc.in ExPASy through Internet.9, 百拇医药
2 Results9, 百拇医药
2.1 Isolation，cloning and sequencing of the PCR product A cDNA fragment about 1 200 bp was amplified with RT-PCR，cloned into pGEM-T easy vector(see Figure 1)and partly sequenced(see Figure 2).
9, 百拇医药Fig 1Isolation and cloning of the cDNA fragment9, 百拇医药
Lane 1.DNA marker：2 000，1 000，750，500，250，1009, 百拇医药
Lane 2.PCR product of angiostatin9, 百拇医药
Lane 3.released insert from recycled TA cloning vector
9, 百拇医药Fig 2The sequence of the cDNA9, 百拇医药
2.2 Analysis of the cDNA The cDNA fragment is not homologous to angiostatin except primer site.There were two incomplete open reading frames within the sequence.The sequences of the encoding peptides were deduced.The correspon-ding peptides are designated peptide 1 and peptide 2 individually.Searching with BLAST and BLASTN in Genbank shows that there are no high homology in terms of nucleotides，but they do have homology in terms of amino acids.The two putative peptides are 21 percent-homologous to large tegument protein of the human herpesvirus 3 and 12 percent-homologous to outer membrane protein rck of Salmonella typhimurium respectively(see figure 3，4)，and are analyzed in Swissprot.
Fig 3Sequence and similarities of peptide 1
g@y, http://www.100md.comFig 4Sequence and similarities of peptide 2g@y, http://www.100md.com
Peptide 1 is about 100 a.a long.It’s molecular weight is 10.823 kD and theoretical PI is 8.88.Since the stop codon in the ORF is within the PCR primer，so we are less sure whether the stop codon is a real or an artificial one.So peptide 1 we deduced from ORF may only be a part of an unidentified protein.It is predicted as a transmembrane protein with the probability of 44.4% and contains a transmembrane helix(from 11 a.a to 29 a.a)with N-terminus inside.The predicted O-glycosylation sites are Thr(No.47 and 65) and Ser(No.51).Peptide 2 is 99 a.a long with molecular weight 11.037 kD and theoretical PI5.29.It’s considered to be a transmembrane protein as well.The transmembrane helix spans from No.2 a.a to 22 a.a with N-terminus inside.Thr(52)is the preferred O-glycosylation site.g@y, http://www.100md.com
3 Discussiong@y, http://www.100md.com
3.1 The cDNA fragment isolated seems ubiquitous in human embryonic liver The cDNA fragment seems ubiquitous in human embryonic liver tissue.It could be isolated from liver samples of three embryos while not from other human tissue samples，such as adult liver，prostate，heart as well as embryonic heart，kidney，lung and brain.It can be inferred that the cDNA fragment was a transi-tory product in the particular stage during the development of embryonic liver.This suggests the possible relation between the expression of the cDNA and the development of liver.
3.2 There are two open reading frames(ORFs)within the cDNA fragment The peptide 1 is homologous to large tegument protein of the human herpesvirus 3.As a transmembrane protein，the prediction of peptide 1 from Swissprot coordinates with large tegument protein.The appearance of large tegument protein is the evidence of viral infection.VAN LOON NM performed direct sequence analysis of polymerase chain reaction-amplified DNA fragments from the large tegument protein(LTP)gene of human herpesvirus 6(HHV-6)with use of uncultured peripheral blood mononuclear cells(PBMCs)from four mother/infant pairs.In two cases，LTP gene sequences were identical in paired mother/infant specimens，thus suggesting that mother-to-infant transmission of HHV-6 may have occurred.The genetic stability of HHV-6 strains was confirmed by the fact that there was no difference between amplified DNA fragments from sequential PBMC samples from two out of two infants analyzed.In contrast ，a change in the amplified viral strain was detected in an infant who had reinfection with HHV-6 variant B(HHV-6B).Furthermore，HHV-6B strains concurrently amplified from saliva and PBMCs from an adult were found to be different.The data suggest that HHV-6 may be frequently transmitted from mother-to-infant and that reinfection with HHV-6B may occur[1].
Part sequence of the peptide 2 is much similar to the outer membrane protein rck(identities 40%)of Salmonella typhimurium.The 17kDa rck is encoded on the Salmonella typhimurium virulence plasmid and confers serum resistance and euka-ryotic cell invasion.The complement resistance mediated by rck is associated with a failure to form fully polymerized tubular membrane attack complexes[2].+4, 百拇医药
The two peptides are both transmembrane proteins with amino termini inside，therefore they can be classified into type Ⅱ membrane proteins.As we know，most transmembrane proteins are receptors and/or signal transduction proteins which are being studied intensively in the world.So the peptides themselves are worthy of further investigation as transmembrane proteins.+4, 百拇医药
3.3 Why are human genes homo-logous to human herpesvirus 3 and Salmonella typhimurium？ The homologous genes between virus and human have been widely accepted as oncogenes.So far we haven’t found any report about existence of human herpesvirus 3 and Salmonella typhimurium homo-logous sequences in the human genome.Why are human genes homologous to microorganisms genes？It is conceivable that genes between virus and animals are homologous because virus genes can integrate into animals’ chromosome by recombination or gain animals’ genes by retrotrans-cription，although whether the virus capture the genes from animals or the virus genes are captured by animals is uncertain.The existence of bacteria-homologous sequence in human genome is a thing hard to explain yet.Since all of the animals and microorganisms develop their genes from the same ancestor called “progenote”[3]，we may reach the explanation of the phenomena in view of evolution someday.The significance of observation reported in this article deserves further research.In addition，other mechanisms by which some genes“flow” between animals and microorganisms might exist as well and inspire us to explore.
3.4 The two ORFs are overlapped Another phenomenon needed mention here is that the two ORFs encoding the peptides homologous to LTP of human herpesvirus 3 and outer membrane protein rck of Salmonella typhimurium are in different reading frames and part of them are overlapped.The overlapping genes were considered to be specific to virus and phage.But recently they have been identified in mammals as well，such as human being[4～7]，rat[8]and mouse[9]etc.Our findings support the evidence that the overlapping genes also exist in human genome.5?, 百拇医药
[Biography] Jiang Ying(1973-)，female，born in Tonghua City， Jilin Province，Posdoc.Ph.D.，field of research：molecular immunology，Tel：(0431)5645911-6462；E-mail：nbumsjc＠public.cc.jl.cn5?, 百拇医药
姜颖(白求恩医科大学免疫学教研室，吉林 长春 130021)5?, 百拇医药
于永利(白求恩医科大学免疫学教研室，吉林 长春 130021)5?, 百拇医药
[REFERENCES]5?, 百拇医药
[1] VAN LOON NM，GUMMULURU S，SHERWOOD DJ，et al.Direct sequence analysis of human herpesvirus 6(HHV-6)sequences from infants and comparison of HHV-6 sequences from mother/infant pairs[J].Clin Infect Dis，1995，21：1 017-1 020.
[2] HEFFERNAN EJ，REED S，HACKETT J，et al.Mechanism of resistance to complement-mediated killing of bacteria encoded by the Salmonella typhimurium virulence plasmid gene rck[J].J Clin Invest，1992，90：953-957.$(rjs, http://www.100md.com
[3] 孙乃恩，孙东旭，朱德煦.分子遗传学[M].南京：南京大学出版社，1996.76-80.$(rjs, http://www.100md.com
[4] SHINTANI S，O’HUIGIN C，TOYOSAWA S，et al.Origin of gene overlap：the case of TCP1 and ACAT2[J].Genetics，1999，152：743-745.$(rjs, http://www.100md.com
[5] BACHMAN NJ，WU W，SCHMIDT TR，et al.The 5’ region of the COX4 gene contains a novel overlapping gene，NOC4[J].Mamm Genome，1999，10：506-512.$(rjs, http://www.100md.com
[6] COOPER PR，SMILINICH NJ，DAY CD，et al.Divergently transcribed overlapping genes expressed in liver and kidney and located in the 11p15.5 imprinted domain[J].Genomics，1998，49：38-51.$(rjs, http://www.100md.com
[7] HERZOG H，DARBY K，BALL H，et al.Overlapping gene structure of the human neuropeptide Y receptor subtypes Y1 and Y5 suggests coordinate transcriptional regulation[J].Genomics，1997，41：315-319.$(rjs, http://www.100md.com
[8] JOSEPH DR.The rat androgen-binding protein(ABP/SHBG)gene contains triplet repeats similar to unstable triplets：evidence that the ABP/SHBG and the fragile X-related 2 genes overlap[J].Steroids，1998，63：2-4.$(rjs, http://www.100md.com
[9] SMITH DP，SPICER J，SMITH A，et al. The mouse Peutz-Jeghers syndrome gene Lkbl encodes a nuclear protein kinase[J].Hum Mol Genet，1999，8：1 479-1 485.$(rjs, http://www.100md.com
[Received date] 1999-11-25； [Revised date] 2000-04-20(姜颖 于永利)