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.

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

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.

Assignment and ordering of twenty-three unique NotI-linking clones containing expressed genes including the guanosine 5'-monophosphate synthetase gene to human chromosome 3.

Twenty-three unique NotI-linking clones, mainly isolated from the NRL1 library, were mapped and ordered by fluorescence in situ hybridization to human chromosome 3. All these clones were partially sequenced around the NotI sites and thus represent sequence-tagged sites. The EMBL nucleotide database was then searched with sequences from the NotI-linking clones using the FASTA program. This search revealed that the NRL-090 clone (at 3q24) contains the gene encoding human guanosine 5'-monophosphate synthetase (GMPS-PEN). To our knowledge, this is the first localization of this gene. Clone NL1-320 (at 3p21.3) contains a gene encoding arginine tRNA (97.3% identity in 73 bp), while clones NRL-063, NRL-097 and NRL-143 contain expressed sequences with unknown functions. Other clones displayed 60-85% similarities to cDNAs, CpG islands and other genes.

A micro-dissection approach for isolation of NotI linking clones from regions frequently deleted in RCC and SCLC.

We demonstrate that micro-dissection can be used for isolating NotI linking clones from the human 3p21-pter region. This approach is an improvement to positional cloning techniques, since NotI linking clones are directly linked with genes.

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.

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.

NotI jumping and linking clones as a tool for genome mapping and analysis of chromosome rearrangements in different tumors.

Long-range restriction site maps are of central importance for mapping the human genome. The use of clones from linking and jumping libraries for genome mapping offers a promising alternative to the laborious procedures used up until now. In the present review, this research field is analyzed with particular emphasis on the implementation of a shot-gun sequencing strategy for genome mapping and the use of NotI linking clones for analysis of rearrangements in tumors and tumor cell lines.

A genome-integrated hepatitis B virus DNA in human neuroblastoma.

An integrated hepatitis B virus (HBV) DNA has been identified in a human neuroblastoma, cloned and sequenced. The integrated HBV DNA is not rearranged, although a 480-bp fragment is deleted. The integrated viral DNA covers the C gene fragment, the region of Pre-S and S, and the 3'-truncated X gene.