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Biomedical subjects

V I Kashuba

Publications and source records attributed to V I Kashuba.

At least 19 recordsLinked to original sources
PubMed2005

High frequency somatic mutations in RASSF1A in nasopharyngeal carcinoma.

High frequency loss of 3p21.3 region is a common event in various kinds of tumors including nasopharyngeal carcinoma (NPC). RASSF1A has been identified as a putative tumor suppressor gene residing in this region. Chromosome alterations and epigenetic changes are commonly observed as mechanisms for inactivation of RASSF1A function. In this study, we applied the PCR-cloning-sequencing strategy to examine somatic mutations in RASSF1A in NPC tissues as compared with the sequences detected in the matched peripheral blood lymphocytes. Our results revealed a high incidence of RASSF1A mutation in primary tumor tissues of NPC. There are totally 35 mutations identified in 74% (17/23) of these NPC cases, including 30 transitions, three transversions and two deletions. Most of these mutations result in amino acid changes: three nonsense (stop codon) mutations, two-1 bp deletion (frameshift), 26 missense and the remaining four are synonymous (silent). No obvious 'hot-spot' mutations were observed in this study. A similarly high rate (74%) of promoter methylation of RASSF1A was also detected in the same group of NPC tissues, but no significant correlation between mutation and methylation was detected. Our results suggest various mechanisms involved in inactivation of RASSF1A function and indicate a critical role of RASSF1A in NPC development.

Adult↗
PubMed2002

Human chromosome 3: integration of 60 NotI clones into a physical and gene map.

Sequence tagged sites generated for 60 NotI clones (NotI-STSs) from human chromosome 3-specific NotI-jumping and NotI-linking libraries were physically located using PCR screening of a radiation hybrid (RH) GeneBridge4 panel. The NotI map of chromosome 3 was generated using these RH-mapping data and those obtained earlier by FISH and sequencing of the corresponding NotI clones. The sequences of the NotI clones showed significant homologies with known genes and/or ESTs for 58 NotI-STSs (97%). These 58 NotI clones displayed 91-100% identity to 54 genes and 23 cDNA/EST clones. One known and two hypothetical protein-coding genes were localized for the first time and nine cDNA clones (unknown genes) were also carefully mapped only in this work. Three newly mapped genes are histone gene H1X (NR1-BK20C) and genes for hypothetical proteins THC1032178 and THC1024604 (NL1-243).

Chromosome Mapping↗
PubMed2001

Initial isolation and analysis of the human Kv1.7 (KCNA7) gene, a member of the voltage-gated potassium channel gene family.

A novel human potassium channel gene was identified and isolated. The maximal open reading frame encodes a protein of 456 amino acids. The predicted product exhibits 91% amino acid identity to the murine voltage-gated potassium channel protein Kv1.7 (Kcna7), which plays an important role in the repolarization of cell membranes. Based on the high similarity, the human gene has been classified as the ortholog of the mouse Kcna7 and given the name Kv1.7 (KCNA7). A structural prediction identified a pore region characteristic of potassium channels and six membrane-spanning domains. Northern expression analysis revealed the gene is expressed preferentially in skeletal muscle, heart and kidney. However, it is expressed at lower level in other tissues, including liver. A single mRNA isoform was observed, with a size of approximately 4.5 kb. Using fluorescence in situ hybridization, the gene was mapped to chromosomal band 19q13.4 (269.13 cR(3000)). A genomic sequence was identified in the database from this region, and the KCNA7 gene structure determined. Computational analysis of the genomic sequence reveals the location of a putative promoter and a likely muscle-specific regulatory region. Initial comparison to the published murine Kcna7 cDNA suggested a different N-terminal sequence for the human protein, however, further analysis suggests that the original mouse sequence contained an error or an unusual polymorphism.

Amino Acid Sequence↗
PubMed1999

Analysis of NotI linking clones isolated from human chromosome 3 specific libraries.

We have partially sequenced more than 1000 NotI linking clones isolated from human chromosome 3-specific libraries. Of these clones, 152 were unique chromosome 3-specific clones. The clones were precisely mapped using a combination of fluorescence in situ hybridization (FISH) and hybridization to somatic cell or radiation hybrids. Two- and three-color FISH was used to order the clones that mapped to the same chromosomal region, and in some cases, chromosome jumping was used to resolve ambiguous mapping. When this NotI restriction map was compared with the yeast artificial chromosome (YAC) based chromosome 3 map, significant differences in several chromosome 3 regions were observed. A search of the EMBL nucleotide database with these sequences revealed homologies (90-100%) to more than 100 different genes or expressed sequence tags (ESTs). Many of these homologies were used to map new genes to chromosome 3. These results suggest that sequencing NotI linking clones, and sequencing CpG islands in general, may complement the EST project and aid in the discovery of all human genes by sequencing random cDNAs. This method may also yield information that cannot be obtained by the EST project alone; namely, the identification of the 5' ends of genes, including potential promoter/enhancer regions and other regulatory sequences

Animals↗
PubMed1999

Identification of new tumor suppressor genes based on in vivo functional inactivation of a candidate gene.

As a step towards developing a new functional test for the identification of tumor suppressor genes, human wild type and mutant RB genes were expressed in the mouse A9 fibrosarcoma cell line under the transcriptional regulation of the tetracycline repressor using two new vectors: pLNCtTA and pETI. Following passage of the transfectants in immunodeficient SCID mice, the wild type RB gene was deleted or functionally inactivated already after the first passage in all 20 tumors tested. In contrast, a non-functional mutant RB gene was maintained in all 10 tumors studied. These results suggest that tests for the identification of tumor suppressor genes may be based on their functional inactivation in vivo, rather than on growth suppression.

Animals↗
PubMed1999

Eukaryotic expression of enzymatically active human immunodeficiency virus type 1 reverse transcriptase.

Reverse transcriptase of human immunodeficiency virus type I is a vitalenzyme in the HIV-1 replication cycle and an attractive target of attempts to arrest a primary viral infection. We designed a vector for eukaryotic expression of the 66 kDa subunit of reverse transcriptase under the control of the immediate early cytomegalovirus promoter. Efficient transient expression of the 66 kDa subunit of reverse transcriptase was achieved in a variety of cells. Immunostaining of the transfected cells revealed the cytoplasmatic localization of reverse transcriptase. Reverse transcriptase activity was detected in all transfected cell lines. Injection of this plasmid encoding the 66 kDa subunit of reverse transcriptase into mice resulted in strong reverse transcriptase-specific immune responses indicating that the 66 kDa subunit of reverse transcriptase is expressed in vivo. Sera from DNA-immunized mice inhibited reverse transcription in vitro.

3T3 Cells↗
PubMed1997

NotI linking/jumping clones of human chromosome 3: mapping of the TFRC, RAB7 and HAUSP genes to regions rearranged in leukemia and deleted in solid tumors.

By applying the 'recognition mask' strategy to 300 mammalian sequences containing NotI sites we demonstrated that 5' ends of genes are highly enriched in NotI sites. A NotI linking clone NL2-252 (D3S1678) containing transferrin receptor (TFRC) gene was used as an initial point for chromosomal jumping. One of the jumping clones, J21-045 traverses 210 kbp and links NL2-252 to NL26 (D3S1632), a NotI linking clone containing highly polymorphic sequences. The TFRC gene was mapped to 3q29, close to the telomeric marker D3S2344, by linkage analysis, a panel of hybrid cell lines, GeneBridge 4 panel and FISH. Clone NLM-007 (D3S4302) was found to contain ras-homologous gene RAB7. By FISH and a panel of hybrid cell lines this gene was mapped to 3q21. This region is of particular interest due to frequent rearrangements in different types of leukemia. Clone L2-081 (D3S4283) containing new member of ubiquitin-specific proteases (HAUSP gene) was localized in 3p21 inspiring further investigation of involvement of this gene in development of lung and renal carcinomas.

Chromosome Mapping↗
PubMed1996

Mapping of 22 Notl linking clones on human chromosome 3 by polymerase chain reaction and somatic cell hybrid panels.

Twenty-two human chromosome 3 derived and partially sequenced Notl linking clones were mapped using two somatic cell hybrid panels. Somatic cell hybrid mapping was performed by Southern hybridization and/or by polymerase chain reaction (PCR), using 300-500 bp CpG-rich sequences surrounding Notl sites. Thus, 22 new Notl site-tagged (sequence tagged sites) STSs were created, distributed over the entire human chromosome 3. The majority of these linking clones tag known or unknown expressed sequences (genes). Together with other physical and genetic mapping methods, localization of Notl linking clones facilitates the construction of a long-range physical map and, at the same time, a transcriptional map of human chromosome 3.

Animals↗
PubMed1996

Mapping of a new MAP kinase activated protein kinase gene (3PK) to human chromosome band 3p21.2 and ordering of 3PK and two cosmid markers in the 3p22-p21 tumour-suppressor region by two-colour fluorescence in situ hybridization.

A Noti-linking clone NL1-210 (D3S1656) that contains the human MAP kinase activated protein kinase (3PK) gene was localized to 3p21.2 on DAPI-banded and propidium iodide (R-bands)-stained chromosomes by fluorescence in situ hybridization (FISH). For more precise localization of 3PK, two cosmid probes were used as a frame. In order to establish this frame, two Noti-linking clones, NL2-008 (D3S1648) and NL3-003 (D3S3872) were used to screen the cosmid library for locus extension. They mapped to 3p21 and were found to belong to two separate contigs of Noti-jumping and linking clones. Using FISH on DAPI-banded metaphase chromosomes, we have determined the precise localization of cosNL2-008 and cosNL3-003 to 3p21.2-p21.1 and 3p22-p21.3 respectively. The 3PK gene was localized to the 3p21.2 region within this frame by two-colour FISH. The orientation of the probes are tel-D3S3872-3PK-D3S1648-cen.

Chromosome Mapping↗
PubMed1996

Human chromosome 3: high-resolution fluorescence in situ hybridization mapping of 40 unique NotI linking clones homologous to genes and cDNAs.

Forty new NotI linking clones representing sequence tagged sites (STSs) were mapped by fluorescence in situ hybridization (FISH) to different regions of human chromosome 3 (HSA3). Clone NL1-245, containing human aminoacylase 1, was localized to 3p21.2-p21.1. Our previous localization of the CLC-2 chloride channel protein gene was refined to 3q27. Clone NL2-316 most likely contains a translocon-associated protein gamma-subunit gene and was mapped to 3q23-q24. To our knowledge, this is the first time this gene has been mapped. One NotI linking clone (NL1-229) probably contains a new protein phosphatase gene. This clone was mapped to 3p25. Five NotI linking clones probably contain human expressed sequence tags (ESTs), as they possess sequences with a high level of identity (> 90%) to cDNA clones. Other clones show 56-85% homology to known mammalian and human genes with various functions, including oncogenes and tumour-suppressor genes. These clones might represent new genes.

Animals↗