[Transcription map of the 13q14 region, frequently deleted in B-cell chronic lymphocytic leukemia patients]. / Transkriptsionnaia karta raiona 13q14, chasto utrachivaemogo u bol'nykh B-kletochnym khronicheskim limfoleikozom.

Deletions in the region located between the STS markers D13S1168 and D13S25 on chromosome 13 are the most frequent genomic changes in patients with B-cell chronic lymphocytic leukemia (B-CLL). After sequencing of this region, two novel candidate genes were identified: C13orf1 (chromosome 13 open reading frame 1) and PLCC (putative large CLL candidate). Analysis of the repeat distribution revealed two subregions differing in composition of repetitious DNA and gene organization. The interval D13S1168-D13S319 contains 131 Alu repeats accounting for 24.8% of its length, whereas the interval GCT16C05-D13S25, which is no more than 180 kb away from the former one is extremely poor in Alu repeats (4.1% of the total length). Both intervals contain almost the same amount of the LINE-type repeats L1 and L2 (20.3 and 21.24%, respectively). In the chromosomal region studied, 29 Alu repeats were found to belong to the evolutionary young subfamily Y, which is still capable of amplifying. A considerable proportion of repeats of this type with similar nucleotide sequences may contribute to the recombinational activity of the chromosomal region 13q14.3, which is responsible for its rearrangements in some tumors in humans.

Comparative sequence analysis of a region on human chromosome 13q14, frequently deleted in B-cell chronic lymphocytic leukemia, and its homologous region on mouse chromosome 14.

Previous studies have indicated the presence of a putative tumor suppressor gene on human chromosome 13q14, commonly deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). We have recently identified a minimally deleted region encompassing parts of two adjacent genes, termed LEU1 and LEU2 (leukemia-associated genes 1 and 2), and several additional transcripts. In addition, 50 kb centromeric to this region we have identified another gene, LEU5/RFP2. To elucidate further the complex genomic organization of this region, we have identified, mapped, and sequenced the homologous region in the mouse. Fluorescence in situ hybridization analysis demonstrated that the region maps to mouse chromosome 14. The overall organization and gene order in this region were found to be highly conserved in the mouse. Sequence comparison between the human deletion hotspot region and its homologous mouse region revealed a high degree of sequence conservation with an overall score of 74%. However, our data also show that in terms of transcribed sequences, only two of those, human LEU2 and LEU5/RFP2, are clearly conserved, strengthening the case for these genes as putative candidate B-CLL tumor suppressor genes.

A cosmid and cDNA fine physical map of a human chromosome 13q14 region frequently lost in B-cell chronic lymphocytic leukemia and identification of a new putative tumor suppressor gene, Leu5.

B-cell chronic lymphocytic leukemia (B-CLL) is a human hematological neoplastic disease often associated with the loss of a chromosome 13 region between RB1 gene and locus D13S25. A new tumor suppressor gene (TSG) may be located in the region. A cosmid contig has been constructed between the loci D13S1168 (WI9598) and D13S25 (H2-42), which corresponds to the minimal region shared by B-CLL associated deletions. The contig includes more than 200 LANL and ICRF cosmid clones covering 620 kb. Three cDNAs likely corresponding to three different genes have been found in the minimally deleted region, sequenced and mapped against the contigged cosmids. cDNA clone 10k4 as well as a chimeric clone 13g3, codes for a zinc-finger domain of the RING type and shares homology to some known genes involved in tumorigenesis (RET finger protein, BRCA1) and embryogenesis (MID1). We have termed the gene corresponding to 10k4/13g3 clones LEU5. This is the first gene with homology to known TSGs which has been found in the region of B-CLL rearrangements.

[Study of alpha-satellite DNA in cosmid libraries, specific for chromosomes 13, 21, and 22, using fluorescence in situ hybridization]. / Issledovanie al'fa-satellitnykh DNK v sostave kosmidnykh bibliotek, spetsifichnykh dlia khromosome 13, 21, i 22, s pomoshch'iu fliuorestsentnoi gibridizatsii in situ.

Fluorescent in situ hybridization (FISH) was employed in mapping the alpha-satellite DNA that was revealed in the cosmid libraries specific for human chromosomes 13, 21, and 22. In total, 131 clones were revealed. They contained various elements of centromeric alphoid DNA sequences of acrocentric chromosomes, including those located close to SINEs, LINEs, and classical satellite sequences. The heterochromatin of acrocentric chromosomes was shown to contain two different groups of alphoid sequences: (1) those immediately adjacent to the centromeric regions (alpha 13-1, alpha 21-1, and alpha 22-1 loci) and (2) those located in the short arm of acrocentric chromosomes (alpha 13-2, alpha 21-2, and alpha 22-2 loci). Alphoid DNA sequences from the alpha 13-2, alpha 21-2, and alpha 22-2 loci are apparently not involved in the formation of centromeres and are absent from mitotically stable marker chromosomes with a deleted short arm. Robertsonian translocations t(13q; 21q) and t(14q; 22q), and chromosome 21p-. The heterochromatic regions of chromosomes 13, 21, and 22 were also shown to contain relatively chromosome-specific repetitive sequences of various alphoid DNA families, whose numerous copies occur in other chromosomes. Pools of centromeric alphoid cosmids can be of use in further studies of the structural and functional properties of heterochromatic DNA and the identification of centromeric sequences. Moreover, these clones can be employed in high-resolution mapping and in sequencing the heterochromatic regions of the human genome. The detailed FISH analysis of numerous alphoid cosmid clones allowed the identification of several new, highly specific DNA probes of molecular cytogenetic studies--in particular, the interphase and metaphase analyses of chromosomes 2, 9, 11, 14, 15, 16, 18, 20, 21-13, 22-14, and X.

[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.

[Subregional localization of recombinant cosmids, containing microsatellite sequences, on human chromosome 13]. / Subregional'naia lokalizatsiia na khromosome 13 cheloveka rekombinantnykh kosmid, soderzhashchikh mikrosatellitnye posledovatel'nosti.

Twenty four recombinant cosmids were subregionally localized by fluorescent in situ hybridization on human chromosomes. Fifteen of the clones were found to belong to only one chromosome: 13 clones located on chromosome 13, one located on chromosome 1, and one on chromosome 11. Nine cosmids were located in nuclear organizer regions. The clones gave signals from NOR regions of chromosome 13 and all other chromosomes containing the NOR region. The cosmid probes were selected from the chromosome 13 cosmid library as ones containing microsatellite repeats with motifs GACA, GACT, GATG, TCC, and CA. Each of the 9 clones located in the NOR region contains microsatellites GACA and TCC. Among the 15 clones giving unique signals, we found 9 clones with the GACT microsatellite, and three clones containing one of the microsatellites GATG, TCC, and CA. These microsatellite-containing clones can be used to make polymorphic genetic markers for fine genetic mapping of chromosome 13.

[Frequency of various mini- and micro-satellite sequences in DNA of human chromosome 13]. / Otsenka chastoty vstrechaemosti nekotorykh mini- i mikrosatellitnykh posledovatel'nostei v DNK khromosomy 13 cheloveka.

The frequency of specific mini- and micro-satellites known also as short tandem repeated sequences (STR) in the human 13 chromosome was estimated by hybridization of STR core oligonucleotides to recombinant cosmid clones transferred to a grid from a human 13 chromosome specific cosmid library ICRF Lawrist 4 C108 (DN L4/HS 13). Oligonucleotides: M13 and Jeffreys minisatellite core sequences and micro-satellite core sequences (TCC)5, (CAC)5, and (GACA)4 were [gamma-32P] end labeled and hybridized to membrane filters carrying good ordered cosmid clones. It was shown that great number of all these mini- and micro-satellite copies (besides of Jeffreys minisatellite) are spread independently along the 13th chromosome. It was also estimated that two or more (GACA)n blocks present in the same cosmid (i.e. on the stretch of 40-50 kb) forming similar groups of clustered micro-satellites. The interesting peculiarity has been recorded that some (GACA)n+ cosmids are also hybridizable to conservative 28SrDNA 3'-fragment that indicates that (GACA)n localization in the nucleoli area. As the result of it we began the creation of a new highly polymorphic markers collections for these chromosome.

[The detection and sequence analysis of the simian T-lymphotrophic retrovirus (STLV-1 Papio) by using the polymerase chain reaction]. / Vyiavlenie i analiz posledovatel'nostei T-limfotropnogo retrovirusa obez'ian (STLV-1 Papio) pri pomoshchi polimeraznoi tsepnoi reaktsii.

The gene amplification technique was used for detection and sequence analysis of STLV-1 Papio proviral DNA. The polymerase chain reaction was performed with a primer pair at tax region of HTLV-1, 7336-7354, sense strand, and 7516-7494, antisense strand. One microgram of DNAs isolated from LUG-4 cells and autopsies was used in a reaction volume of 50 microliters involving 30 cycles of amplifications. The reaction product was blunt-end cloned into pUC19 cut with Smal. The sequence was done with T7-polymerase using 32P-dATR as a label. Our results indicate that STLV-1 Papio provirus is actually present in the cells of a lymphoid cell line and tumor cells of lymphomatous monkeys. There are some differences between STLV-1 Papio and reported sequences of HTLV-1 and STLV-1.

[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.