The gene from the short arm of chromosome 3, at D3F15S2, frequently deleted in renal cell carcinoma, encodes acylpeptide hydrolase.

Loss or inactivation of a gene on the short arm of chromosome 3 may contribute to the genesis of renal cell carcinoma. A gene that corresponds to the most frequently lost RFLP site (D3F15S2) is expressed in a variety of human tissues, and at a particularly high level in the kidney. Its expression is markedly reduced in renal cell carcinoma. A database search showed that the gene product is closely related to or identical with acylpeptide hydrolase. The nucleotide identity between the rat acylpeptide hydrolase and the human gene at D3F15S2 is 88%, compatible with normal species differences. It is therefore likely that the human gene product is acylpeptide hydrolase. The renal cell carcinoma is then associated with a decrease of acylpeptide hydrolase activity. The gene may represent a tumor suppressor gene, whose loss contributes to the development of renal cell carcinoma. It might be speculated that it could act e.g. by affecting the activity of a small acetylated growth factor. Alternatively, its decreased expression may merely reflect the impairment of differentiation in RCC, compared to normal kidney. Loss of a linked but irrelevant gene by the 3p deletion is another possibility.

An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends.

For the preparation of gene libraries, DNA from lambda EMBL3 phage was digested with SalI and EcoRI, and the cohesive ends partially filled-in by addition of dTTP, dCTP and Klenow fragment of DNA polymerase I (PolIk). Genomic DNA was cleaved partially with Sau3A and subsequently incubated with dATP, dGTP and PolIk. The phage and genomic DNAs were then mixed and ligated. The recombinant DNAs were packaged in vitro. The efficiency of packaging was 10(5)-10(6) of infectious phage lambda particles per microgram of the genomic DNA (as compared to approx. 10(7) per microgram for the wild-type lambda DNA). This procedure is very rapid and requires only microgram quantities of genomic DNA for preparing an entire gene library. The other important advantage is that multiple independent insertions of genomic DNA cannot occur in a single recombinant phage and self-ligation of phage DNA is blocked. It is also applicable for other SalI-containing vectors.

Family of human Na+, K+-ATPase genes. Structure of the gene for the catalytic subunit (alpha III-form) and its relationship with structural features of the protein.

The primary structure of a gene of the Na+, K+-ATPase multigenic family in the human genome has been determined. The gene corresponds to a hypothetical alpha III-form of the enzyme catalytic subunit. The gene comprises over 25,000 bp, and its protein coding region includes 23 exons and 22 introns. Possible correlation between structural features of the protein and location of introns in the gene are discussed.

The family of human Na+,K+-ATPase genes. No less than five genes and/or pseudogenes related to the alpha-subunit.

Five different nucleotide sequences have been found in the human genome homologous to the gene of the alpha-subunit of Na+,K+-ATPase. A comparative analysis of the primary structure of these genes in the region 749-1328 (in coordinates of cDNA from the pig alpha-subunit) is presented.

[Vectors for constructing representative genome libraries]. / Vektory dlia konstruirovaniia predstavitel'nykh genomnykh klonotek.

The general methodology for constructing genomic libraries of different types of vectors is discussed. Various ways of selection against non-recombinant molecules in generated libraries are considered. The general features of well-known vectors (lambda EMBL3, lambda EMBL4) and of new ones (lambda Ch40, lambda SK5, lambda FIX, pWE and other) are presented. A special attention is paid to vectors lambda SK9 and SK18 that have the features of lambda and M13 phages and of plasmids (diphasmids). Data on phasmids (lambda pMYF131 and lambda pSL51) and hyphages MC18 and MC19 are also presented.

[Construction of libraries of structural genes (cDNA) and "jumping" gene libraries in lambda vectors]. / Konstruirovanie klonotek strukturnykh genov (kDNK) i "pryzhkovykh" klonotek v lambda-vektorakh.

Main approaches and methods used for constructing "jamping" and cDNA libraries, including novel ones, that omit the employment of methylases and linkers, are presented. The advantages and drawbacks of the well-known and new lambda vectors, suitable for the purposes mentioned, are discussed. Special attention is paid to diphasmids lambda ZAP, lambda SK12, lambda SK15, that combine features of lambda and M13 phages and of plasmids. The convenience and difficulties of "jumping" libraries for physical mapping of chromosomes are briefly considered.

[Construction of a gene library using partial filling of DNA sticky ends]. / Poluchenie klonotek genov s ispol'zovaniem chastichnoi dostroiki lipkikh kontsov DNK.

To prepare gene libraries, the incomplete filling of protruding ends has been used. DNAs from phages EMBL 3 and EMBL 3a were sequentially digested with SalI and EcoRI, followed by addition of dTTP, dCTP, and DNA polymerase I (Klenow's fragment). Separately, a genomic DNA was partially cleaved with Sau3AI, followed by addition of dATP, dGTP, and Klenow's fragment. The fragmented phage and genomic DNAs were mixed and ligated, and the recombinant DNAs packed in vitro with the phage proteins. The effectiveness of packaging per microgram of genomic DNA was 10(5) to 10(6) (for the wild phage DNA, 10(7)). The proposed procedure is very rapid and needs only microgram quantities of genomic DNA for preparing a representative gene library. It is also useful for other vectors, containing SalI sites.

[Nucleotide sequence of the rplL gene coding for ribosomal protein L7/L12 of Pseudomonas putida]. / Nukleotidnaia posledovatel'nost' gena rplL, kodiruiushchego ribosomnyi belok L7/L12 Pseudomonas putida.

The Pseudomonas putida rpl L gene coding for ribosomal protein L7/L12 was cloned and sequenced. Although Asp55 residue in L7/L12 was previously shown to be conservative in ten different organisms, the Pseudomonas putida L7/L12 proved to contain Asn55, thus showing that Asp55 is not invariant.

[Genes coding for RNA-polymerase in bacteria. II. Conservative sites in the central region of the beta-subunit of Pseudomonas putida RNA-polymerase]. / Geny, kodiruiushchie RNK-polimerazu bakterii. II. Konservativnye uchastki v tsentral'noi chasti beta-sub''edinitsy RNK-polimerazy Pseudomonas putida.

SalI--L fragment of the P. putida rpoBC operon has been sequenced and conservative regions of the central part of the RNA-polymerase beta-subunit have been determined. Amino and acid residues interacting with Zn2+ are postulated.

[Genes encoding bacterial RNA-polymerase. I. Cloning of rpoBC-operon of Pseudomonas putida and its physical map]. / Geny, kodiruiushchie RNK-polimerazu bakterii. I. Klonirovanie rpoBC-operona Pseudomonas putida i ego fizicheskaia karta.

The P. putida rpoBC operon, coding for beta and beta' subunits of RNA polymerase, was cloned and its physical map constructed.

[Bank of sequences specific for human genome. I. The structure of one of the repeats in the Alu family]. / Poluchenie banka posledovatel'nostei, spetsifichnykh dlia genoma cheloveka. I. Struktura odnogo iz povtorov Alu-semeistva.

A bank enriched in sequences specific for the human genome was obtained. In course of the analysis, a clone containing an Alu family repeat was identified and its primary structure determined.

NotI linking clones as a tool for joining physical and genetic maps of the human genome.

To study the connection among NotI linking clones, CpG islands, and genes, the sequence surrounding 143 NotI sites was determined. These NotI linking clones were isolated from human chromosome 3-specific libraries and contain an average C + G content of 65%. These clones represent sequence-tagged sites that can be positioned onto chromosome maps and used for generating a long-range NotI map of the human genome. A majority (about 90%) of these clones contain transcribed sequences, as detected by Northern blot hybridization, providing an efficient link between physical and functional (genetic) maps. The GenBank nucleotide database was searched with sequences from these NotI linking clones. For many clones, homology was found to human and other vertebrate genes. About 20 clones contained various repeats in their sequences and may represent microsatellite loci. Most of these NotI linking clones therefore represent evolutionarily conserved DNA fragments and also can be used for comparative genome mapping of other mammalian species. In addition, approximately 20% of all sequenced human CpG island-containing genes and more than 12% of all well-characterized human genes were found to possess NotI restriction sites. This is at least 2-5 times more than has been previously estimated and suggests that NotI sites have a much stronger association with genes.

New strategy for mapping the human genome based on a novel procedure for construction of jumping libraries.

A novel procedure for construction of jumping libraries is described. The essential features of this procedure are as follows: (1) two diphasmid vectors (lambda SK17 and lambda SK22) are simultaneously used in the library construction to improve representativity, (2) a partial filling-in reaction is used to eliminate cloning of artifactual jumping clones and to obviate the need for a selectable marker. The procedure has been used to construct a representative human NotI jumping library (220,000 independent recombinant clones) from the lymphoblastoid cell line CBMI-Ral-STO, which features a low level of methylation of its resident EBV genomes. A human chromosome 3-specific NotI jumping library (500,000 independent recombinant clones) from the human chromosome 3 x mouse hybrid cell line MCH 903.1 has also been constructed. Of these recombinant clones 50-80% represent jumps to the neighboring cleavable NotI site. With our previously published method for construction of linking libraries this procedure makes a new genome mapping strategy feasible. This strategy includes the determination of tagging sequences adjacent to NotI sites in random linking and jumping clones. Special features of the lambda SK17 and lambda SK22 vectors facilitate such sequencing. The STS (sequence tagged site) information obtained can be assembled by computer into a map representing the linear order of the NotI sites for a chromosome or for the entire genome. The computerized mapping data can be used to retrieve clones near a region of interest. The corresponding clones can be obtained from the panel of original clones, or necessary probes can be made from genomic DNA by PCR.

Localization of human ARF2 and NCK genes and 13 other NotI-linking clones to chromosome 3 by fluorescence in situ hybridization.

Two human genes containing NotI sites, ADP-ribosylation factor (ARF2) and melanoma NCK protein, were mapped by fluorescence in situ hybridization to 3p21.2-->p21.1 and 3q21, respectively. Thirteen other NotI-linking clones, representing sequence tagged sites, were also mapped to different regions of human chromosome 3. Two of these clones that contain sequences 80% homologous to the rat tropoelastin gene and brain Cl- channel protein CLC-2 gene probably represent new human genes closely related to the known rat genes.