Spectrum of mutations in BRCA1 gene in hereditary forms of breast and ovarian cancer in Russian families.

The 5382insC mutation predominated (94%) in the spectrum of detected mutations of BRCA1 gene. High incidence of this mutation in familial breast cancer detected for the first time attested to origination of 5382insC mutation from the European part of Russia. The percentage of families with mutations in BRCA1 gene and familial predisposition to ovarian cancer was significantly higher than in hereditary predisposition to breast cancer (p<0.007). These data suggest that clinical manifestation of the mutation depends on genotypical factors other than the position of this mutation in BRCA1 gene. The results prompt screening for hereditary predisposition to these diseases.

[TUB9 polymorphism in the CFTR gene of cystic fibrosis patients, carriers, and healthy donors from the Moscow region. SSCP and restriction analyses]. / Polimorfizm TUB9 v gene TRBM bol'nykh mukovistsidozom, nositelei i zdorovykh donorov Moskovskogo regiona. SSCP-analiz i restriktsionnyi analiz.

Data on the screening of 266 non-delta F508 chromosomes (42 cystic fibrosis patients, 43 carriers, and 48 healthy donors from the Moscow region) for the presence of structural abnormalities within the tenth exon of the CFTR gene conducted by means of the single-stranded conformation polymorphism (SSCP) technique in nonisotope modification are presented. The method used made it possible to detect three SSCP variants, one of which was present in cystic fibrosis patients (23.8%) and carriers (9.3%), but not in healthy donors. Sequencing of the 5 amplified DNA fragments carrying this SSCP variant revealed an A-->G substitution in the 1525-61 position, which indicated the presence of TUB9 polymorphism with allele 1 in the homozygous state in all cases tested. The three SSCP variants described corresponded to the three allelic variants of TUB9 polymorphism as judged by MnlI restriction analysis of the amplified tenth exon sequence. The modified SSCP technique is also suitable for routine screening for the G542X, G55ID, and W1282X point mutations within the CFTR gene. The frequency distribution of polymorphic TUB9 marker alleles across the non-delta F508 chromosomes in the three studied groups were estimated. Homozygotes for the TUB9 allele 1 were shown to have identical GATT-TUB9-M470V haplotypes.

[Cosmid libraries containing DNA from human chromosome 13]. / Issledovanie kosmidnykh bibliotek, soderzhashchikh DNK khromosomy 13 cheloveka.

We characterized two cosmid libraries constructed from flow-sorted chromosome 13 at the Imperial Cancer Research Fund (ICRF), UK (13,000 clones) and Los Alamos National Laboratory (LANL), USA (17,000 clones). After storage for two years, clones showed high viability (95%) and structural stability. EcoR I and Hind III restriction patterns were studied in more than 500 ICRF and 200 LANL cosmids. The average size of inserts was shown to be 35-37 kb in both the libraries. Most cosmids (83% and 93% of ICRF and LANL libraries, respectively) exceed the lower size limit of DNA fragments that can be packaged and represent a good source for physical mapping of chromosome 13. Total length of inserts is four and five genome equivalents in the ICRF and LANL libraries, respectively. ICRF cosmids showed hybridization to 22 of 24 unique probes tested, which corresponds to a 90% probability of having any DNA fragment represented in the library. More than 1 Mb of chromosome 13 is overlapped by 90 cosmids of 22 groups revealed. A chromosomal region of more than 150 kb, containing the ATP1AL1 gene for alpha-1 peptide of Na+, K(+)-ATPase, is covered by 12 cosmids forming a contig. The results of restriction and hybridization analyses are stored in a CLONE database. These data and all the cosmids described are publicly available.

[Determination and analysis of the primary structure of a genomic sequence adjacent to the 3'-end of the human tissue plasminogen activator gene]. / Opredelenie i analiz pervichnoi struktury genomnoi posledovatel'nosti, prilegaiushchei k 3'-kontsu gena tkanevogo aktivatora plazminogena cheloveka.

Primary structure was determined for the recently cloned f1/BglII-fragment [19] containing 2102 b.p. of the human tissue plasminogen activator (tPA) gene 3' end and adjacent DNA region. Computer analysis has revealed an Alu-repeat 820 b.p. downstream the tPA gene; the sequence proved to have a considerable homology (86-88%) with the Alus from the 3'-untranslated regions (3'UTRs) of cytochrome P-450, lysozyme and p53 protein human mRNAs. The same homology was estimated for this Alu in reversed orientation and Alus from the 3'UTRs of some other human mRNAs. In contrast, the homology between this 3' end tPA gene flanking Alu-repeat and other Alus dispersed throughout the gene introns either direct or reversed, was less than 70%. The polyadenylation signal AATAAA downstream the Alu and two nearby signals CACAG and GTGTT resembling consensus sequences CACAG and YGTGTTYY, respectively, were also detected. The two latter motifs located close to the 3' ends in most mammalian genes are likely to regulate mature mRNA formation. The comparison of the sequenced spaser flank adjacent to the tPA gene with short homologous sequence from the same genomic region primary structure reported previously has revealed discrepancies (substitutions, deletions or insertions) in 21 nucleotide positions. The nucleotide sequence of E. coli uvrB gene fragment (980 b.p.) is also reported. This E. coli gene fragment was cloned accidentally within the f1/BglII-fragment being an artifact of the host-vector system used.

[Isolation and characteristics of DNA fragments for the region of the tissue plasminogen activator genes and areas adjacent to it in the human genome]. / Vydelenie i kharakteristika fragmentov DNK oblasti gena tkanevogo aktivatora plazminogena i prilegaiushchikh k nemy uchastkov genoma cheloveka.

Fragments overlapping the tPA gene and its 5'- and 3'-flanking regions were isolated from human liver DNA library cloned in lambda Charon4A vector. A BglII fragment comprising the 3' end and the adjacent genomic region (total length 3.7 kb) was subcloned in plasmid pUC19 and its restriction map was determined. The nucleotide sequence of the 5' region of this fragment was compared with the 3' end region of the tPA gene and the corresponding regions of five published variants of tPA mRNA cDNA from different tissues; discrepancies in seven positions were revealed, which might be caused by intragenomic polymorphism.

[Characteristics of the pH2-42 probe for the D13S25 locus of human chromosome 13: nucleotide sequence, localization, and PCR markers]. / Kharakteristika proby pH2-42 lokusa D13S25 13-i khromosomy cheloveka: nukleotidnaia posledovatel'nost', lokalizatsiia, PTsR-markery.

The sequence of the HindIII-HindIII fragment of probe pH2-42 of locus D13S25 of human genome is given. Localization of the probe in q14-q21 of human chromosome 13 is confirmed by hybridization in situ. Seven oligonucleotide primers for the polymerase chain reaction are chosen so that amplified products almost completely cover the analyzed sequence. Reconstruction of localization of polymorphic SspI-sites in D13S25 was based on the data of Bowcock and Hebert [3] and this study. The results obtained make it possible to use the primer sets to screen cosmid libraries and to mark the D13S25 locus of human chromosome 13.

[Stable maintenance of dicentric mini-chromosomes in CHL4 mutants in yeast]. / Stabil'noe podderzhanie ditsentricheskikh mini-khromosom u mutanta drozhzhei po genu chl4.

Earlier we have identified the chl4-1 mutation in a screen for yeast mutants with increased loss of chromosome III and circular artificial minichromosome in mitosis. Mutation in the CHL4 gene leads to a 50-100-fold promotion in the rate of chromosome loss per cell division compared to the isogenic wild type strain. Detailed analysis of behaviour of the circular minichromosome marked by the CUP1 gene has shown that minichromosome nondisjunction (2:0 segregation) leading to an increase in the copy number of minichromosome in part of a cell population is the main reason of minichromosome instability in the mutant. The unique peculiarity of chl4-1 mutation is the ability of the strains carrying this mutation to stably maintain circular dicentric minichromosomes without any rearrangement during many generations. (In the wild type strains dicentric minichromosomes are extremely unstable. As a consequence of that there is a strong selection for cells harboring monocentric derivatives in a population of cells derived from a cell containing a dicentric plasmid). Introduction of the second centromere into one of the natural chromosomes (chromosomes II or III) in the chl4-1 mutant leads to the same dramatic consequences as that in the wild type strain (mitotic lag of cells harboring dicentric chromosomes and, as a result of that, selective pressure for cells harboring monocentric derivatives of dicentric chromosome). A genomic clone of CHL4 was isolated by complementation of the chl4-1 mutation. Nucleotide sequence analysis of CHL4 revealed a 1.4-kb open reading frame with a predicted 53-kDa protein sequence. Analyzing the sequence of the CHL4 protein we have found a region meeting the necessary requirements for the helix-turn-helix (HTH) structure. This region of the CHL4 protein has about 40% homology with the repressor of tryptophane operon (TrpR) of E. coli. A strain containing a null allele of CHL4 was viable under standard growth conditions, but had temperature-sensitive phenotype (conditional lethality at 34 degrees C). We suggest that the CHL4 gene product is one of the components of the segregation cell machinery.

[CHL15--a new gene controlling the replication of chromosomes in saccharomycetes yeast: cloning, physical mapping, sequencing, and sequence analysis]. / CHL15--novyi gen, kontroliruiushchii replikatsiiu khromosom u drozhzhei--sakharomitsetov: klonirovanie, fizicheskoe kartirovanie, sekvenirovanie i funktsional'nyi analiz.

We have analyzed the CHL15 gene, earlier identified in a screen for yeast mutants with increased loss of chromosome III and artificial circular and linear chromosomes in mitosis. Mutations in the CHL15 gene lead to a 100-fold increase in the rate of chromosome III loss per cell division and a 200-fold increase in the rate of marker homozygosis on this chromosome by mitotic recombination. Analysis of segregation of artificial circular minichromosome and artificially generated nonessential marker chromosome fragment indicated that sister chromatid loss (1:0 segregation) is a main reason of chromosome destabilization in the chl15-1 mutant. A genomic clone of CHL15 was isolated and used to map its physical position on chromosome XVI. Nucleotide sequence analysis of CHL15 revealed a 2.8-kb open reading frame with a 105-kD predicted protein sequence. At the N-terminal region of the protein sequences potentially able to form DNA-binding domains defined as zinc-fingers were found. The C-terminal region of the predicted protein displayed a similarity to sequence of regulatory proteins known as the helix-loop-helix (HLH) proteins. Data on partial deletion analysis suggest that the HLH domain is essential for the function of the CHL15 gene product. Analysis of the upstream untranslated region of CHL15 revealed the presence of the hexamer element, ACGCGT (an MluI restriction site) controlling both the periodic expression and coordinate regulation of the DNA synthesis genes in budding yeast. Deletion in the RAD52 gene, the product of which is involved in double-strand break/recombination repair and replication, leads to a considerable decrease in the growth rate of the chl15 mutant. We suggest that CHL15 is a new DNA synthesis gene in the yeast Saccharomyces cerevisiae.

[son Pseudogenes do not contain five repeating elements of the region of complete tandem repeats present in the homologous sequence of the son gene]. / Psevdogen son ne soderzhit piati povtoriaiushchikhsia élementov oblasti sovershennykh tandemnykh povtorov, prisutstvuiushchikh v gomologichnoi posledovatel;nosti gena son.

Recently we have published a sequence of the coding region of the son gene, containing at least six areas of the tandem repeats [V.V. Bliskovsky, F.B. Berdichevsky, A.V. Tkachenko, M.E. Belova, I.M. Chumakov--Molecularnaya biologiya, 1992, V. 26. P. 793-806; V.V. Bliskovsky, A.V. Kirillov, V.M. Zacharyev, I.M. Chumakov--Molecularnaya biologiya, 1992, V. 26. P. 807-812]. The presence of several areas of tandem repeats with different nucleotide sequences of the repeated elements within one and the same gene supports the proposition that genomic localization of the sequence influences its duplication. Here we present a nucleotide sequence of the son pseudogene isolated as the result of hybridization screening of a human genomic library using the sequence of the son gene as a probe. The comparison of the gene and the pseudogene nucleotide sequences shows that the sequence of pseudogene does not contain five repeated units of an area of the tandem repeats, which are presented in the homology sequence of the son gene. Because the pseudogene was probably generated by the reverse son-gene transcript insertion to the genome, and so the nucleotide sequences of the coding region of the gene and the sequence of the pseudogene were identical at this moment, the differences between the gene and the pseudogene are the results of their evolution after the generation of the pseudogene. Possible factors, influenced the son gene and the son pseudogene evolution are discussed.