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1.
Endogenous retrovirus-related sequences exist within the normal genomic DNA of all eukaryotes, and these endogenous sequences have been shown to be important to the nature and biology of related exogenous retroviruses and may also play a role in cellular functions. To date, no endogenous sequences related to human immunodeficiency virus type 1 (HIV-1) have been reported. Herein we describe the first report of the presence of nucleotide sequences related to HIV-1 in human, chimpanzee, and rhesus monkey DNAs from normal uninfected individuals. We also present the isolation and characterization of two of these endogenous HIV-1-related sequences, EHS-1 and EHS-2. With use of low-stringency Southern blot hybridization, complex banding patterns were detected in human DNA with 5' and 3' HIV-1-derived probes. When an HIV-1 env region probe was used, we detected a less complex, conserved banding pattern in human DNA as well as a related but distinct banding pattern in chimpanzee and rhesus monkey DNAs. EHS-1 and -2 were cloned from normal human genomic DNA libraries by using the env region probe. Clone EHS-1 shows sequence similarity with the domain of the envelope cellular protease cleavage site of HIV-1, while EHS-2 has sequence similarity to the overlapping reading frame for Rev and gp41. Stringent hybridization of EHS-1 back to primate genomic DNA indicates two distinct EHS-1 loci in normal human DNA, an identical band pattern in chimpanzee DNA, and a single locus in rhesus monkey DNA. Likewise, EHS-2 is present as a single highly conserved locus in all three species. An oligonucleotide derived from EHS-2 across a region of near identity to HIV-1 detects a complex banding pattern in all primates tested similar to that seen with the 3' HIV-1 probe. These data suggest that most of the HIV-1-related sequences identified in primate DNA share a common core of nucleic acid sequence found in both EHS-2 and rev and that some of these HIV-1-related sequences have additional larger regions of sequence similarity to HIV-1.  相似文献   

2.
Regardless of the route of transmission, it is generally accepted that the human immunodeficiency virus type 1 (HIV-1) quasispecies transmitted from an infected individual to an uninfected individual is genetically homogeneous. This finding and the observation that HIV-1 genotypes in recipients are minor variants in the donors suggest strongly that selection for specific variants occurs. However, most analyses have been limited to the V3 region of env. In addition, the exact time at which most new infections occurred was not known, making it almost impossible to analyze virus populations present in donor-recipient pairs at the time of HIV-1 transmission. To circumvent this problem, three chimpanzees were inoculated with a genetically defined stock of cell-free HIV-1/JC499 by one of three routes: intravenously or via the cervical or penile mucosa. PCR products of the C2-to-V5 region of env were amplified from both proviral DNA and virion RNA in blood samples collected soon after infection and were screened by heteroduplex analysis (HDA). Those PCR products with distinct HDA banding patterns were cloned and sequenced. In all three animals, transmitted variants encoded one of two V3-loop populations identified in the inoculum, indicating relative homogeneity in this region. However, different virus populations, defined by combinations of specific V4 and V5 sequences, were found when variants in the animal inoculated intravenously (at least 13 V4-plus-V5 combinations) were compared with those in the two animals inoculated by the mucosal routes (limited to only four V4-plus-V5 combinations). The only V4-plus-V5 population in variants found in all three chimpanzees was the major population in the inoculum, which contained viruses with more than 30 different V4-plus-V5 combinations. That the majority of the V4-plus-V5 genotypes in variants transmitted to all three animals were minor populations in the inoculum indicated that selective transmission defined by the V4-plus-V5 regions had occurred but that distinct populations were transmitted by parenteral versus mucosal routes. These results indicate that the putative homogeneity of HIV-1 variants in a newly infected individual might be an artifact of the region of the env gene evaluated and that regions other than V3 might play a major role in selective transmission.  相似文献   

3.
Viral RNA was extracted from plasma samples collected from five individuals during the period of viremia before seroconversion in primary infection with human immunodeficiency virus type 1 (HIV-1) and amplified by polymerase chain reaction. Nucleotide sequence analysis of amplified DNA from the V3 and V4 hypervariable regions indicated that the initial virus population of each acutely infected individual was completely homogeneous in sequence. No intrasample variability was found among the 44,090 nucleotides sequenced in this region of env, contrasting with the high degree of variability normally found in seropositive individuals. Paradoxically, substantial sequence variability was found in the normally high conserved gag gene (encoding p17) in most of the preseroconversion samples. The diversity of p17 sequences in samples that were homogeneous in V3 and V4 can most readily be explained by the existence of strong selection for specific env sequences either upon transmission or in the interval between exposure and seroconversion in the exposed individual. Evidence that localizes the selected region upon transmission to V3 is provided by the similarity or identity of V3 loop sequences in five individuals with epidemiologically unrelated HIV-1 infections, while regions flanking the V3 loop and the V4 hypervariable region were highly divergent. The actual V3 sequences were similar to those associated with macrophage tropism in primary isolates of HIV, irrespective of whether infection was acquired by sexual contact or parenterally through transfusion of contaminated factor VIII. Proviral DNA sequences in peripheral blood mononuclear cells remained homogeneous in the V3 and V4 regions (and variable in p17gag) for several months after seroconversion. The persistence of HIV sequences in peripheral blood mononuclear cells identical to those found at primary infection in the absence of continued virus expression provides an explanation for the previously observed differences in the composition of circulating DNA and RNA populations in sequential samples from seropositive individuals.  相似文献   

4.
The evidence for genetic variation can be traced to Mendel's experiments: The discovery of the laws of heredity was made possible by the expression of segregating alleles. Since that time, the study of genetic variation in natural populations has been characterized by a gradual discovery of ever-increasing amounts of genetic variation. In the early decades of this century geneticists thought that an individual is homozygous at most gene loci and that individuals of the same species are genetically almost identical. Recent discoveries suggest that, at least in outcrossing organisms, the DNA sequences inherited one from each parent are likely to be different for nearly every gene locus in every individual; ie, that every individual may be heterozygous at most, if not all, gene loci. But the efforts to obtain precise estimates of genetic variation have been thwarted for various reasons.  相似文献   

5.
In the NL4-3 strain of human immunodeficiency virus type 1 (HIV-1), regulatory elements responsible for the relative efficiencies of alternative splicing at the tat, rev, and the env/nef 3' splice sites (A3 through A5) are contained within the region of tat exon 2 and its flanking sequences. Two elements affecting splicing of tat, rev, and env/nef mRNAs have been localized to this region. First, an exon splicing silencer (ESS2) in NL4-3, located approximately 70 nucleotides downstream from the 3' splice site used to generate tat mRNA, acts specifically to inhibit splicing at this splice site. Second, the A4b 3' splice site, which is the most downstream of the three rev 3' splice sites, also serves as an element inhibiting splicing at the env/nef 3' splice site A5. These elements are conserved in some but not all HIV-1 strains, and the effects of these sequence changes on splicing have been investigated in cell transfection and in vitro splicing assays. SF2, another clade B virus and member of the major (group M) viruses, has several sequence changes within ESS2 and uses a different rev 3' splice site. However, splicing is inhibited by the two elements similarly to NL4-3. As with the NL4-3 strain, the SF2 A4b AG dinucleotide overlaps an A5 branchpoint, and thus the inhibitory effect may result from competition of the same site for two different splicing factors. The sequence changes in ANT70C, a member of the highly divergent outlier (group O) viruses, are more extensive, and ESS2 activity in tat exon 2 is not present. Group O viruses also lack the rev 3' splice site A4b, which is conserved in all group M viruses. Mutagenesis of the most downstream rev 3' splice site of ANT70C does not increase splicing at A5, and all of the branchpoints are upstream of the two rev 3' splice sites. Thus, splicing regulatory elements in tat exon 2 which are characteristic of most group M HIV-1 strains are not present in group O HIV-1 strains.  相似文献   

6.
Population-genetic studies have been remarkably productive and successful in the last decade following the invention of PCR technology and the introduction of mitochondrial and microsatellite DNA markers. While mitochondrial DNA has proven powerful for genealogical and evolutionary studies of animal populations, and microsatellite sequences are the most revealing DNA markers available so far for inferring population structure and dynamics, they both have important and unavoidable limitations. To obtain a fuller picture of the history and evolutionary potential of populations, genealogical data from nuclear loci are essential, and the inclusion of other nuclear markers, i.e. single copy nuclear polymorphic (scnp) sequences, is clearly needed. Four major uncertainties for nuclear DNA analyses of populations have been facing us, i.e. the availability of scnp markers for carrying out such analysis, technical laboratory hurdles for resolving haplotypes, difficulty in data analysis because of recombination, low divergence levels and intraspecific multifurcation evolution, and the utility of scnp markers for addressing population-genetic questions. In this review, we discuss the availability of highly polymorphic single copy DNA in the nuclear genome, describe patterns and rate of evolution of nuclear sequences, summarize past empirical and theoretical efforts to recover and analyse data from scnp markers, and examine the difficulties, challenges and opportunities faced in such studies. We show that although challenges still exist, the above-mentioned obstacles are now being removed. Recent advances in technology and increases in statistical power provide the prospect of nuclear DNA analyses becoming routine practice, allowing allele-discriminating characterization of scnp loci and microsatellite loci. This certainly will increase our ability to address more complex questions, and thereby the sophistication of genetic analyses of populations.  相似文献   

7.
Investigation of human immunodeficiency virus type 1 (HIV-1) in the genital tract of women is crucial to the development of vaccines and therapies. Previous analyses of HIV-1 in various anatomic sites have documented compartmentalization, with viral sequences from each location that were distinct yet phylogenetically related. Full-length RNA genomes derived from different compartments in the same individual, however, have not yet been studied. Furthermore, although there is evidence that intrapatient recombination may occur frequently, recombinants comprising viruses from different sites within one individual have rarely been documented. We compared full-length HIV-1 RNA sequences in the plasma and female genital tract, focusing on a woman with high HIV-1 RNA loads in each compartment who had been infected heterosexually and then transmitted HIV-1 by the same route. We cloned and sequenced 10 full-length HIV-1 RNA genomes from her genital tract and 10 from her plasma. We also compared viral genomes from the genital tract and plasma of four additional heterosexually infected women, sequencing 164 env and gag clones obtained from the two sites. Four of five women, including the one whose complete viral sequences were determined, displayed compartmentalized HIV-1 genomes. Analyses of full-length, compartmentalized sequences made it possible to document complex intrapatient HIV-1 recombinants that were composed of alternating viral sequences characteristic of each site. These findings demonstrate that the genital tract and blood harbor genetically distinct populations of replicating HIV-1 and provide evidence that recombination between strains from the two compartments contributes to rapid evolution of viral sequence variation in infected individuals.  相似文献   

8.
Genes of the major histocompatibility complex (MHC) have been characterized in all extant lineages of mammals. The tammar wallaby (Macropus eugenii) is well established as a model marsupial species; however, no classical MHC sequences have been described from this species. We have isolated two MHC class II -chain sequences from a tammar wallaby spleen cDNA library using a tammar MHC class II probe. These sequences belong to the marsupial MHC class II DBB gene family. Two additional DBB sequences were amplified from tammar wallaby genomic DNA. All four sequences were obtained from the same individual, indicating that there are at least two DBB loci in the tammar wallaby.Nucleotide sequence data reported are available in the GenBank database under the accession numbers AY438038–AY438042  相似文献   

9.
BACKGROUND AND AIMS: Crassula hunua and C. ruamahanga have been taxonomically controversial. Here their distinctiveness is assessed so that their taxonomic and conservation status can be clarified. METHODS: Populations of these two species were analysed using morphological, chromosomal and DNA sequence data. KEY RESULTS: It proved impossible to differentiate between these two species using 12 key morphological characters. Populations were found to be chromosomally variable with 11 different chromosome numbers ranging from 2n = 42 to 2n = 100. Meiotic behaviour and levels of pollen stainability were both variable. Phylogenetic analyses showed that differences exist in both nuclear and plastid DNA sequences between individual plants, sometimes from the same population. CONCLUSIONS: The results suggest that these plants are a species complex that has evolved through interspecific hybridization and polyploidy. Their high levels of chromosomal and DNA sequence variation present a problem for their conservation.  相似文献   

10.
The human and simian immunodeficiency viruses encode at least six gene products that apparently serve regulatory functions. To evaluate the regulation of simian immunodeficiency virus gene expression at the level of RNA splicing, we used the polymerase chain reaction to amplify and clone cDNAs corresponding to a large array of mRNAs from infected cells. We identified mRNAs that used splice acceptor sites upstream of the initiator codons for tat, rev, vpr, nef, vif, and vpx, suggesting that these proteins may be expressed from different mRNAs. We also provide hybridization data suggesting that the same splice acceptor site may be used for both rev and env mRNAs. Furthermore, we isolated both tat and rev cDNAs that utilized three alternative splice acceptor sites at the start of coding exon 2, indicating that different versions of these proteins may be encoded. Finally, approximately 10 to 20% of simian immunodeficiency virus mRNAs spliced an intron from their untranslated 5' ends, and sequences contained within this intron constituted a portion of the tat-responsive TAR element. Thus, alternative pre-mRNA splicing adds a level of complexity to simian immunodeficiency virus expression, which may affect several levels of gene regulation.  相似文献   

11.
HLA-G is a nonclassical class I MHC molecule of unknown function expressed on human trophoblast. The level of polymorphism at the HLA-G locus is of considerable importance, since the paternally inherited gene product is exposed to the maternal immune system during pregnancy. However, previous studies of HLA-G polymorphism using genomic DNA samples have produced conflicting results. Our aim was to investigate polymorphism in trophoblast HLA-G mRNA from pregnancies in ten Caucasian and twelve Afro-Caribbean women by RT-PCR. A similar PCR protocol was also applied to umbilical cord blood genomic DNA from two Caucasian and two Afro-Caribbean neonates. Caucasian cDNA yielded only two different sequences: G*01011, and one containing a previously reported synonymous substitution. Afro-Caribbean samples yielded these sequences as well as one previously reported conservative (leucine-to-isoleucine) substitution. PCR amplification from genomic DNA samples from both populations using previously published primer pairs generated sequences containing multiple substitutions, many of which were nonsynonymous. More than two sequences were produced from genomic DNA from each individual. In contrast, amplification from the same genomic DNA using new primers complementary to exons of the HLA-G gene yielded the same few sequences generated from cDNA. These results suggest that polymorphism at the HLA-G locus is extremely limited in Caucasian and Afro-Caribbean populations. This suggests that spurious polymorphism has been reported in African Americans due to the use of intron-complementary PCR primers on genomic DNA samples. The monomorphic nature of HLA-G may allow trophoblast to carry out the immunological functions of class I-bearing tissues without compromising successful pregnancy.  相似文献   

12.
We analyzed virus sequences in two monkeys infected with SIVmac239 and two monkeys infected with SHIVnef that maintained high, persisting viral loads. Sequence changes were observed consistently at four loci in all four animals: a single nucleotide change in the Lys-tRNA primer binding site in the 5' long terminal repeat; two nucleotide changes that resulted in two amino acid changes in the pol gene product; and a single nucleotide change in the region of the simian immunodeficiency virus genome where the rev and env genes overlap, resulting in changes in the predicted amino acid sequences of both gene products. None of these mutations were seen in short-term cultures of CEMx174 cells infected with SIVmac239 or SHIVnef. At all four positions in all four animals, the new sequences represented consensus sequences for primate lentiviruses, whereas the inoculum sequences at these four loci have either never been or rarely been reported outside of SIVmac239. Thus, although cloned SIVmac239 is consistently pathogenic and consistently induces high viral load set points, it is clearly less than optimal at these four nucleotide positions.  相似文献   

13.
Using DNA heteroduplex tracking assays, we characterized human immunodeficiency virus type 1 env V4/V5 genetic populations in multiple blood plasma samples collected over an average of 7 months from 24 chronically infected human subjects. We observed complex and dynamic V4/V5 genetic populations in most subjects. Comparisons of V4/V5 and V1/V2 population changes over the course of the study showed that major shifts in genetic populations frequently occurred in one region but not the other, and these observations were independently confirmed in one subject by single-genome sequencing. These results suggest that the V1/V2 and V4/V5 regions of env often evolve independently during chronic infection.  相似文献   

14.
Miller HC  Lambert DM 《Molecular ecology》2004,13(12):3709-3721
The Chatham Island black robin, Petroica traversi, is a highly inbred, endangered passerine with extremely low levels of variation at hypervariable neutral DNA markers. In this study we investigated variation in major histocompatibility complex (MHC) class II genes in both the black robin and its nonendangered relative, the South Island robin Petroica australis australis. Previous studies have shown that Petroica have at least four expressed class II B MHC genes. In this study, the sequences of introns flanking exon 2 of these loci were characterized to design primers for peptide-binding region (PBR) sequence analysis. Intron sequences were comprised of varying numbers of repeated units, with highly conserved regions immediately flanking exon 2. Polymerase chain reaction primers designed to this region amplified three or four sequences per black robin individual, and eight to 14 sequences per South Island robin individual. MHC genes are fitness-related genes thought to be under balancing selection, so they may be more likely to retain variation in bottlenecked populations. To test this, we compared MHC variation in the black robin with artificially bottlenecked populations of South Island robin, and with their respective source populations, using restriction fragment length polymorphism analyses and DNA sequencing of the PBR. Our results indicate that the black robin is monomorphic at class II B MHC loci, while both source and bottlenecked populations of South Island robin have retained moderate levels of variation. Comparison of MHC variation with minisatellite DNA variation indicates that genetic drift outweighs balancing selection in determining MHC diversity in the bottlenecked populations. However, balancing selection appears to influence MHC diversity over evolutionary timescales, and the effects of gene conversion are evident.  相似文献   

15.
16.
The dry rot fungus Serpula lacrymans is a devastating basidiomycete occurring in wooden constructions in temperate regions worldwide. In this study, we compare the genetic structures of two invasive populations from Europe and Japan. Microsatellite data from 14 loci and DNA sequences from four loci demonstrated that the two populations were highly differentiated. Significant isolation by distance effect was observed in Europe and Japan. Higher genetic variation was observed within the Japanese population than within the European population, corresponding with the observed higher richness of vegetative compatibility types in Japan, indicating that there has been a higher level of gene flow from the Asian source populations to Japan than to Europe. The European population is genetically more homogenous with only six detected vegetative compatibility types. Various tests indicate that both the European and the Japanese populations have gone through population bottlenecks prior to population expansion. No identical multi‐locus genotypes were observed within Japan and very few within Europe, indicating limited clonal dispersal. Deviations from Hardy Weinberg expectations were observed both in Europe and Japan and heterozygote excess were observed at several loci, especially in Europe. Possible explanations for this pattern are discussed.  相似文献   

17.
We constructed five chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIVMAC) and four SIVMAC mutants by recombinant DNA techniques. Three chimeric clones and all mutants with an alteration in either the vif, vpx, vpr, or nef gene were infectious to human CD4-positive cell lines. The susceptibility of macaque monkey peripheral blood mononuclear cells (PBMC) to infection by these mutants and chimeras was examined in vitro. Macaque PBMC supported the replication of wild-type and vpx, vpr, and nef mutant SIVMAC strains. A chimera carrying the long terminal repeats (LTRs), gag, pol, vif, and vpx of SIVMAC and tat, rev, vpu, and env of HIV-1 was also replication competent in PBMC. In contrast, HIV-1, the vif mutant of SIVMAC, a chimera containing rev and env of SIVMAC, and a chimera containing vpx, vpr, tat, rev, and env of SIVMAC did not grow in PBMC. Western immunoblotting analysis of the replicating chimera in PBMC confirmed the hybrid nature of the virus. These data strongly suggested that the sequence important for macaque cell tropism lies within the LTR, gag, pol, and/or vif sequences of the SIVMAC genome.  相似文献   

18.
The study of expression of several human immunodeficiency virus type 1 proviral mutants in human cells in the presence or absence of rev (trs/art) protein reveals that rev increases the levels of unspliced and env mRNA and the accumulated structural viral proteins. rev protein produced from appropriate expression vectors fully complements the rev-defective mutants. rev requires the presence of a specific cis-acting sequence for its function. This rev-responsive element sequence has been localized within a 520 base-pair fragment in the env region of human immunodeficiency virus type 1. gag and env expression is coordinately regulated by rev. Two independent cis-acting elements localized in the gag and env regions are responsible for the low levels of gag and env mRNA in the absence of rev. These elements are different than the rev-responsive element and act independent of each other.  相似文献   

19.
The murine leukemia virus (MuLV) sequence associated with the resistance allele of the Fv-4 gene (Fv-4r) was molecularly cloned from genomic DNA of uninfected mice carrying this allele. The 5.2-kilobase cloned EcoRI DNA fragment (pFv4) was shown by nucleotide sequencing to contain 3.4 kilobases of a colinear MuLV-related proviral sequence which began in the C-terminal end of the pol region and extended through the env region and the 3' long terminal repeat. Cellular sequences flanked the 3' as well as the 5' ends of the truncated MuLV sequence. Alignment of the N-terminal half of the pFv4 env sequence with ecotropic, mink cell focus-forming, and xenotropic MuLV env sequences established the relatedness of pFv4 and ecotropic MuLV env sequences. A subcloned 700-base pair segment (pFv4env) from the 5' env region of pFv4 was used as an Fv-4-specific probe; it hybridized specifically to the Fv-4r-associated proviral sequence but not to endogenous ecotropic MuLV proviral DNA under high stringency. All Fv-4-resistant mice contained the same retroviral segment associated with the same flanking cellular DNA. Expression of Fv-4r-specific mRNA was demonstrated in the spleens of Fv-4r mice but not Fv-4s mice, supporting the previously proposed resistance model based on interference.  相似文献   

20.
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