Isolation of chromosome 21-specific yeast artificial chromosomes from a total human genome library.

A new approach for the isolation of chromosome-specific subsets from a human genomic yeast artificial chromosome (YAC) library is described. It is based on the hybridization with an Alu polymerase chain reaction (PCR) probe. We screened a 1.5 genome equivalent YAC library of megabase insert size with Alu PCR products amplified from hybrid cell lines containing human chromosome 21, and identified a subset of 63 clones representative of this chromosome. The majority of clones were assigned to chromosome 21 by the presence of specific STSs and in situ hybridization. Twenty-nine of 36 STSs that we tested were detected in the subset, and a contig spanning 20 centimorgans in the genetic map and containing 8 STSs in 4 YACs was identified. The proposed approach can greatly speed efforts to construct physical maps of the human genome.

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 lambda 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.

The coding region of the human c-mos pseudogene contains Alu repeat insertions.

We have determined the nucleotide sequence of an 841-bp fragment derived from a segment of the human genome previously cloned by Chumakov et al. [Gene 17 (1982) 19-26] and Zabarovsky et al. [Gene 23 (1983) 379-384] and containing regions homologous to the viral mos gene probe. This sequence displays homology with part of the coding region of the human and murine c-mos genes, contains several termination codons, and is interrupted by two Alu-family elements flanked by short direct repeats. Probably, the progenitor of the human c-mos gene was duplicated approximately at the time of mammalian divergence, was converted to a pseudogene, and acquired insertions of two Alu elements.

The expression of skin-specific gene K51 in the epidermal layer of human skin and in basal cell carcinoma cells.

Gene K51 probe isolated previously from the rat genomic library has been used to study the expression of its human counterpart by in situ hybridization and Northern blot analysis. A polyA-containing transcript of human gene K51 of 3 kb size has been detected in embryonic skin. The gene is also expressed in the epidermis of newborn humans and adults, but not in the adjacent mesenchymal tissues. Immunostaining with keratin antisera revealed predominantly earlier stage expression of K51 than cytokeratin markers. Sebaceous and sweat glands also contain cells expressing K51 gene. K51 expression was found in the cells of eight individual basal cell carcinomas tested, with the level of expression lower than in keratinocytes from normal human epidermis. We propose that K51 gene expression could serve as a convenient marker for the study of the process of skin keratinocyte development and the changes in this process associated with skin cancers and dysplasia.

Human genome: proto-oncogenes and proretroviruses.

A brief review of the studies undertaken at the Laboratory for Molecular Bases of Oncogenesis (Institute of Molecular Biology, Moscow) till middle of 1984 is presented. The human genome contains multiple dispersed nucleotide sequences related to the proto-oncogene mos and to proretroviral sequences in tight juxtaposition to each other. From sequencing appropriate cloned fragments of human DNA in phage and plasmid vectors it follows that one of these regions, NV-1, is a pseudogene of proto-mos with partial duplications and two Alu elements intervening its coding sequence, and the other, CL-1, seems to be also a mos-related gene with a deletion of the internal part of the structural gene. CL-1 is flanked by a proretroviral-like sequence including tRNAiMet binding site and U5 (part of the long terminal repeat). The proretroviral-like sequences are transcribed in 21-35S poly(A)+RNA abundant in normal and malignant human cells. Two hypotheses are proposed: endogenous retroviruses take part in amplification of at least some proto-oncogenes; proto-oncogenes are inactivated via insertion of movable genetic elements and conversion into pseudogenes. Potential oncogenicity of a normal human genome undergoes two controversial influences: it increases due to proto-oncogene amplification and decreases due to inactivation of some of them.

[Decoding of the primary structure of the son3 region in human genome: identification of a new protein with unusual structure and homology with DNA-binding proteins]. / Rasshifrovka pervichnoi struktury uchastka son3 genoma cheloveka: identifikatsiia novogo belka, imeiushchego neobychnuiu strukturu i gomologiiu s DNK-sviazyvaiushchimi belkami.

From the human embryonic cDNA library a son3 transcript was cloned and sequenced (1454 base pairs). Determination of its sequence revealed only one open reading frame, whereas five other frames contained a number of termination codons. Translation of the son3 sequence in the unique open reading frame into the amino acid sequence by computer and comparison with the NBRF protein data bank showed that the son3 fragment codes for a new previously unknown polypeptide with the following properties: a) it contains a cluster of short tandemly arranged repeats 7-12 amino acid in length located in the middle part of son3; b) it comprises a region homologous to DNA binding structural proteins (for example, gallin, 55%) and to regulatory proteins coded by the family of proto-oncogene myc; c) it comprises a region homologous to the oncoprotein coded by proto-oncogene mos (human, murine).

[Nucleotide sequence of the fragment of transcriptionally active locus in rat genome and analysis of a putative protein product]. / Nukleotidnaia posledovatel'nost' uchastka transkriptsionno aktivnogo lokusa K51 genoma krysy i analiz predpolagaemogo belkovogo produkta.

Part of the transcriptionally active rat genomic locus, K51, has been sequenced (2,321 b.p.). The coding DNA chains was identified and two long open reading frames (ORF) were found by computer analysis one of which seems to be much less probable. By transcriptional mapping, at least two exons (A - about 110 b.p., B - 549 b.p.) were revealed. Computer search showed that the deciphered nucleotide and amino acid sequence was absent in the data-banks up to the end of 1988. Numerous GC rich direct repeats were noticed in the fragment. At protein level, resemblance with hypervariable N- and C-terminal domains of cytokeratins was visualized. Some regions of the putative protein product possess relatedness with some other gene products, particularly with ATP binding domains of mos oncoprotein.

[Tissue specificity of expression of k51 gene in rodents]. / Tkanevaia spetsifichnost' ékspressii kena k51 u gryzunov.

A new method for studding gene expression--the histoblotting was used for the detection of k51 gene expression in rodent tissues. We have demonstrated several advantages of this method over in situ hybridization for the study of gene expression in epitelial tissues in particular. The transcription of the new gene k51 was detected in the upper layers of mouse and rat skin during epidermis development before and after rat birth. These data were confirmed by Northern hybridization analysis. We found that the expression of k51 occurs mainly in flat multilayer epithelium and is not simply connected with the degree of days of rat embryo development; it is high in newborn skin and lowers during hair growth. The expression of k51 can serve as a marker of a certain stage of adult skin development.

[Identification of a protein product of a novel human gene SON and the biological effect upon administering a changed form of this gene into mammalian cells]. / Identifikatsiia belka-produkta novogo gena cheloveka SON i biologichekii éffekt pri vvedenii izmenennoi formy étogo gena v kletki mlekopitaiushchikh.

We have investigated the functional activity of human son gene, that possesses the homology to mos and myc genes. Specific antibodies (antiserum) were raised to synthetic peptide, that corresponds to son-protein 943-963 amino acid residues. With this antiserum the presence of son-protein was showed in lysates of cultured human cells transformed by adenovirus type 5, RAT 2 cells and primary human embryonic fibroblasts. son-Protein molecular weight (92 kDa) was determined by the method of electrophoresis in SDS-polyacrylamide gel. Thus, it was shown the presence of son gene protein in animal and human cells. To determine a possible son gene role in mammalian cells we have cloned the 3' part (2667 b.p.) of son cDNA in retroviral vector pPS-3-neo. Transformed cells of different lines were selected. A large portion of this cells changed their morphology. New protein product (120 k), that reacted with antiserum to son specific peptide, was found together with p92son in these clones.

[Evolutionary variants of the mos gene]. / Evoliutsionnye varianty gena mos.

The occurence of members of mos oncogene family in the vertebrates genome has been studied with the help of highly labeled single-stranded DNA probes. These included subgenic v-mos clones as well as the unique sequence--specific recombinants from mos-related human locus gp5 and the K51 locus from rat genome. The probe from gp5 (mos pseudogene) interacts only with DNA of primates and of rodents. On the other hand, mos gene and the gene from K51 locus are present in all vertberates tested. Recent duplication of the main mos gene in Artiodactyla and Perissodactyla orders of mammals was identified. The persistence of K51 and mos genes during evolution indicates their importance. The segregation of three mos-related genes in human-hamster hybrids points to their location on different human chromosomes.

[Regions of human genome-containing analogs of oncogenes and retroviral genes. II. A new class of retrovirus-like elements]. / Uchastki genoma cheloveka, soderzhashchie analogi onkogenov i genov retrovirusov. II. Novyi klass retrovirusopodobnykh élementov.

Earlier we have found that recombinant phage lambda gp5 contains the sequences homologous to v-mos oncogene and retroviruses. After the nucleotide sequence determination we have found the region with homology to U5 part of retroviral LTR. Adjacent to this region are sequences complementary to 3'-end of tRNAMet. Numerous transcripts reacting with subcloned U5 probe from gp5 are present in polyadenylated RNA fraction from human cells. The humane genome contains several copies of these region, with two of them residing in gp5 locus. On the basis of these data we deduced the presence in the human genome of a new class of retroviral-like elements, existing probably as part of new human endogeneous retrovirus (HuEV).

[Regions of human genome containing analogs of oncogenes and retrovirus genes. I. A family of c-mos genes and unusual structure of ORA-gp5 locus]. / Uchastki genoma cheloveka, soderzhashchie analogi onkogenov i genov retrovirusov. I. Semeistvo genov c-mos i neobychnaia struktura lokusa ORA-gp5.

The structural organization of a number of recombinant phages previously selected from the human gene library has been studied. On the basis of comparison of physical maps and hybridization to cloned probes it was deduced that different human loci with the homology to v-mos are represented in lambda recombinants. The physical map of the cloned region of the human genome designated as ORA-gp5 was constructed. The sequences of three different genetical elements v-mos-related oncogene, mammalian type C retrovirus and Alu type repeat are interspersed in this structure. The hypothesis concerning the probable origin of this locus has been proposed. The mosaical structure of ORA-gp5 could be the result of the integration of mammalian retrovirus in the vicinity to c-mos gene with subsequent recombination and transposition. The resulting potentially oncogenic structure was later inactivated by the integration of Alu-type repeats.

[Segments of the human genome, containing analogs of oncogenes and retrovirus genes. 4. Primary structure of the mos-related AO section of the ORAgp5 locus]. / Uchastki genoma cheloveka, soderzhashchie analogi onkogenov i genov retrovirusov. 4. Pervichnaia struktura mos-rodstvennogo uchastka AI lokusa ORAgp5.

AO region (884 b.p.) of ORAgp5 locus has been sequenced and proved to contain mos-related regions, as well as fragments structurally similar to proretroviral elements and Alu repeats. The data obtained are in accordance with earlier hypotheses on retroviral involvement in mos gene family generation and the role of Alu repeat insertion in the inactivation of mos-related genes. Molecular hybridisation studies showed the structural conservation of the segment in ORAgp5 locus comprising the AO region among higher primates. These data indicate that AO region of ORAgp5 was formed not later than 65-70 MYR ago and that the present structure of this region was kept during last 50 MYR.

[Regions of the human genome containing homologs of the oncogenes and retrovirus genes. 3. The primary structure of the mos-related region CL-1 from the ORAgp5 locus]. / Uchastki genoma cheloveka, soderzhashchie analogi onkogenov i genov retrovirusov. 3. Pervichnaia struktura mos-rodstvennogo uchastka CL-1 iz lokusa ORAgp5.

The nucleotide sequence of 686 bp from the cloned human genome locus gp5 has been determined. Analysis of this sequence has revealed a statistically significant homology with both the viral and murine mos genes. The region of mos homology contains two adjacent homologous domains, whereas their counterparts in viral mos gene are separated by 471 bp. The position of mos homologous region in the close vicinity to LTR of endogenous human viral-like repeat is in accordance with the hypothesis of retroviral involvement in the process of mos gene amplification.

[Transcription-active locus of the human genome from a gene family]. / Transkriptsionno-aktivnyi lokus genoma cheloveka iz semeistva genov.

Previously described genomic sequences, related to viral oncogene mos (human mos pseudogene gp5 and main c-mos gene), are either totally silent or show highly limited degree of expression in some specialized tissues. The mos-related clone K51 isolated previously from rat genome was used here to study expression of the gene comprised. We have demonstrated the presence of abundant transcripts in rat and human brain, lung, skin and muscle. The pattern of transcription changes after the birth and is shown to depend on the presence of higher molecular weight transcripts in adult tissues. With the help of hybridization to K51 probe, we have isolated three clones from human embryonic cDNA library. One of these (son3) also hybridizes to the viral mos gene. These results evidence strongly in favour of the hypothesis that K51 recombinant contains a new member (son) of mos-related family of oncogenes, with the broad spectrum of tissue specificity.

[Expression in mouse L cells of mos-related ORAgp5 locus cloned from the human genome]. / Ekspressiia v L-kletkakh myshi mos-rodstvennogo lokusa ORAgp5, klonirovannogo iz genoma cheloveka.

The recombinant locus ORAgp5 containing the regions homologous to protooncogene mos was previously cloned from the human genome. In order to monitor the expression of this human genome region, we have transfected the recombinant phage gp5 into mouse LMtk cells. One of these transfectants contained several copies of gp5. ORAgp5 sequences were found to be expressed in 1.5 kb RNA from this clone.

[Expression of gene K51 in normal epidermis and various epithelial skin tumors in man]. / Ekspressiia gena K51 v normal'nom épidermise i nekotorykh épitelial'nykh opukholiakh kozhi cheloveka.

The expression of gene K51 in the cells of human normal epidermis and epithelial skin tumors was investigated using in situ hybridization method with radioactive probe. The K51 gene transcripts were detected in the epidermis, sebaceous and sweat glands of human embryo and adult skin. The level of gene expression was higher in the stratum granulosum than in the basal layer of the skin. K51 gene expression was also found in the basal cell and metatypical carcinomas, with the level of expression lower than in the neighbouring epidermis and higher than in the surrounding skin stromal cells. Thus, K51 gene is expressed in the skin epidermis of human embryo and adults but the level of its activity is dramatically decreased in the cells of skin epithelial tumors. This potentially may be important as a diagnostic test.

[Use of sedimentation under conditions of acidic denaturation for the determination of molecular weights of RNAs]. / Primenenie sedimentatsii v usloviiakh kislotnoi denaturatsii dlia opredeleniia molekuliarnogo vesa RNK

The conditions for acidic denaturation of double stranded RNA were found. Under these conditions a limited degradation of high molecular weight viral RNA took place. This degradation was determined by the degree of fragmentation and loss of infectivity at acidic conditions. It was found that acidic denaturation of RNA in the solutions of low ionic strength was accompanied by a considerable increase of sedimentation coefficient. Under these conditions the coefficients of sedimentation and molecular weights of RNAs studied are connected by the following function S20=2.84-10(-2) Mr0.689. The conclusion has been drawn that the sedimentation under the conditions for acidic denaturation could be used both for molecular weight determination and the practical preparation of unaggregated strands of RNA.

Cloning the human major histocompatibility complex in YACs.

To clone the human major histocompatibility complex (MHC), 53 YACs, with an average size of 490 kb, were isolated and characterized from the CEPH YAC library. These YACs were organized in a single large contig covering more than 4000 kb. Furthermore, a complete physical map of the previously uncloned HLA class I region was established from partial and/or total digestions of 15 YACs spanning 2000 kb. This resulted in the establishment of the first YAC contig that spans the entire MHC region and constitutes an essential step in the isolation of all of the genes present in the region.