Vertebrate DNAs contain nucleotide sequences related to the transforming gene of avian myeloblastosis virus.

Avian myeloblastosis virus contains a continuous sequence of approximately 1,000 nucleotides which may represent a gene (amv) responsible for acute myeloblastic leukemia in chickens. This sequence appears to have been acquired from chicken DNA and to be substituted for the envelope gene in the viral genome. We used hybridization probes enriched for the amv sequences and conditions that facilitate annealing of partially homologous nucleotide sequences to show that cellular sequences related to amv are present in the genomes of all vertebrates ranging from amphibians to humans but were not detected in fish, sea urchins, or Escherichia coli. In contrast to the preceding findings, nontransforming endogenous proviral nucleotide sequences closely related to the remainder of the avian myeloblastosis virus genome and to the entire myeloblastosis-associated helper virus are present only in chicken DNA. The amv-related cellular sequences appear to be highly conserved during evolution and to be contained at only one or a few locations in the genome of vertebrates. Within closely related species, they appear to share common evolutionary genetic loci. These findings and similar ones obtained with other highly oncogenic retroviruses containing a transforming gene suggest a general mechanism for acquisition of viral oncogenic sequences and an essential role for these sequences in the normal cellular state.     

Nucleotide sequence and distinctive characteristics of the env gene of endogenous feline leukemia provirus.

Nucleotide sequence analysis of the env gene of two different endogenous feline leukemia virus (FeLV) loci, CFE-6 and CFE-16, of domestic cats revealed the following characteristics. (i) Both proviruses contain an open reading frame in the env region; (ii) whereas the full complement of the exogenous FeLV env is generally present in CFE-6 DNA, it is truncated in CFE-16 DNA such that the 5' half of the gp70 domain and the untranslated region 3' to the p15E domain have been fused by an internal deletion, resulting in loss of the C-terminal half of the gp70- and all of the p15E-coding sequences; (iii) endogenous env is highly homologous to large sequence domains conserved in all three exogenous FeLV subgroups (A, B, and C) but is similar to FeLV-B sequence domains in the variable regions detected in these viruses; and (iv) there are four other sequence domains, one residing at the C terminus of gp70 and three scattered in p15E, which are unique for the endogenous env, thereby distinguishing it from the FeLV-B gene.     

A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene.

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.     

Nonsporulating bacterial species contain DNA sequences homologous to the Bacillus spore-specific C-protein gene.

Genes for small, acid-soluble spore proteins (SASPs) are ubiquitous among the spore-forming bacteria and are expressed only during sporulation. Although they perform the function of amino acid storage in spores, the members of the SASP-C multigene family probably serve additional functions, so that similar sequences might be present in non-spore-formers. Using the SASP-C gene (ssp-c) as a hybridization probe, restriction digests of whole genomic DNA from seven nonsporulating bacterial species were examined for similar sequences. Hybridization was found in four species: Streptococcus pyogenes, Staphylococcus aureus, Neisseria sicca, and Mycobacterium phlei, indicating the presence of similar sequences in some, but not all, of the non-spore-formers. In each of these positive species, multiple bands hybridized. A 4.5-kb hybridizing fragment from S. pyogenes and a 9.0-kb hybridizing fragment from M. phlei have been cloned and partially sequenced. These fragments show substantial DNA sequence homology to ssp-c and their deduced amino acid sequences show substantial homology to SASP-C.     

Novel human endogenous sequences related to human immunodeficiency virus type 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.     

The bovine and ovine genomes contain multiple sequences homologous to the alpha-lactalbumin-encoding gene

Bovine and ovine (pseudo)genes homologous to the alpha-lactalbumin-encoding gene are described. In both cases, sequence analysis reveals homology extending downstream from exon 2. Southern analysis indicates the presence of a family of alpha-lactalbumin-related sequences in the bovine genome.     

Chicken erythrocyte polynucleosomes which are soluble at physiological ionic strength and contain linker histones are highly enriched in beta-globin gene sequences.

Mature chicken erythrocyte polynucleosomes which are soluble at physiological ionic strength are enriched in beta-globin DNA sequences. Vitellogenin chromatin, which is not expressed in this tissue, is found in aggregation prone, salt insoluble chromatin. There is a direct correlation between the size of soluble fragments and the degree of globin gene enrichment, with the largest fragments being most highly enriched. The highly globin enriched (about 50 fold) polynucleosomes contain significantly elevated levels of acetylated histones H4, H2A.Z, and H2B, and ubiquitinated (prefix "u") histones H2A and H2B (with a significant relative increase of uH2B over uH2A). These polynucleosomes were complexed with histones H1 and H5 but at a lower level than that found in unfractionated chromatin.     

Use of gene transfer and a novel cosmid rescue strategy to isolate transforming sequences.

Mouse Lewis Lung tumor DNA was ligated to a cosmid containing a geneticin (G418)/kanamycin resistance gene and transferred into NIH3T3 cells. Recipient cells were first selected for geneticin resistance and subsequently for their ability to grow as a tumour when injected into nude mice. By repeating this transfection procedure with DNA from resultant tumours, geneticin-resistant NIH3T3 cells were obtained which were tumorigenic and contained approximately 1-5 copies of the transferred cosmid. The functional oncogene was cloned by preparing cosmid libraries of third round tumour DNAs, using a cosmid which does not contain a kanamycin resistance gene. Due to the original linkage of the oncogene with the cosmid containing the kanamycin resistance gene, a series of kanamycin-resistant cosmids were isolated, five of which contained an active oncogene. Subsequent analysis showed that the oncogene present was highly related to the human N-ras gene. Using a DNA probe from the MLL N-ras gene, a non-transforming counterpart was isolated from mouse liver DNA. A comparison between the two N-ras genes showed that a mutation at the amino acid position corresponding to 61 in the human gene is responsible for transforming activity of the rescued gene.     

Does the genome of Corylus avellana L. contain sequences homologous to the self-incompatibility gene of Brassica?

Self-incompatibility is a genetic mechanism enforcing cross-pollination in plants. Hazelnut (Corylus avellana L.) expresses the sporophytic type of self-incompatibility, for which the molecular genetic basis is characterized only in Brassica. The hypothesis that the hazelnut genome contains homologs of Brassica self-incompatibility genes was tested. The S-locus glycoprotein gene (SLG) and the kinase-encoding domain of the S-receptor kinase (SRK) gene of B. oleracea L. were used to probe blots of genomic DNA from six genotypes of hazelnut. Weak hybridization with the SLG probe was detected for all hazelnut genotypes tested; however, no hybridization was detected with PCR-generated probes corresponding to two conserved regions of the SLG gene. One of these PCR probes included the region of SLG encoding the 11 invariant cysteine residues that are an important structural feature of all S-family genes. The present evidence suggests that hazelnut DNA hybridizing to SLG differs significantly from the Brassica gene, and that the S-genes cloned from Brassica will not be useful for exploring self-incompatibility in hazelnut.     

Human nucleotide sequences related to the transforming gene of a murine sarcoma virus: studies with cloned viral and cellular DNAs

A recombinant plasmid, pI26, has been constructed by cloning into pBR322 a transforming gene of murine sarcoma virus (a Moloney strain, clone 124, MSV) synthesized by detergent-treated virions. From this plasmid a XbaI-HindIII fragment has been isolated which contains only mos-specific sequences. This mos-specific probe has been used for screening a human gene library cloned in bacteriophage λ Charon 4A. Of these, 19 clones have been isolated containing mos-related sequences. By physical mapping and molecular hybridization it has been shown that these sequences are neighboured by DNA regions related to Moloney murine leukemia virus. Recombinant phages have also been found containing human inserts related to MLV, not to the mos gene. The possible existence of murine-like endogenous retroviruses in the normal human genome, including that of a sarcoma type, is discussed. By Northern blotting, expression of the cellular c-mos gene has been detected in mouse liver treated with a hepatocarcinogen. The general significance of the suggested model for evaluating the relationship between chemical carcinogenesis and oncogene expression is discussed.     

Human papillomavirus type 6 long control region and human cellular DNA contain related sequences.

We have identified a region of human papillomavirus type 6 (HPV-6) DNA that hybridizes with human cellular DNA containing no detectable HPV DNA sequences. The region of hybridization has been localized to a segment of the viral long control region between the end of the L1 open reading frame and the late polyadenylation signal and is likely contained within a 94-base-pair insertion at nucleotide 7350 which is present in the cloned HPV-6b DNA used for these studies. Restriction fragments of HPV-6 DNA from seven patients suggested that this insert was present in these naturally occurring viral genomes as well. The presence of this insert was confirmed by direct sequence analysis of polymerase chain reaction-amplified segments from four naturally occurring HPV-6 genomes. By analogy with other systems, this insert and surrounding sequences may function to destabilize the HPV-6 late mRNA.     

Cosuppression of nonhomologous transgenes in Drosophila involves mutually related endogenous sequences.

Cosuppression refers to the phenomenon in which silencing among dispersed homologous genes occurs. Here we demonstrate that two nonhomologous reciprocal fusion genes, white-Alcohol dehydrogenase (w-Adh) and Adh-w, exhibit cosuppression using the endogenous Adh sequence as an intermediary. Deletion of the endogenous Adh gene eliminates the interaction, while reintroduction of an 8.6 kb Adh fragment restores the silencing. Using truncated Adh constructs, a nontranscribed segment in the Adh regulatory region was found to be one of the sequences required for homology recognition. The silencing interaction is initiated during early development. The silenced transgenes are associated with the Polycomb group complex of chromatin proteins.     

Absence from the mouse genome of DNA sequences related to the globin gene family.

Radioactively labelled DNA, complementary to mouse α and β globin messenger RNA, was annealed with unlabelled mouse embryo DNA under conditions of both optimum and lowered stringency. Although an increase in saturation of unlabelled DNA fragments with complementary DNA molecules is produced by lowered stringency, the values obtained are within the range expected for the known globin chains.It is concluded that within the limits of our experimental conditions, there does not exist a large family of DNA sequences related to the globin genes.