Rinat Gizatullin

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.

Human cell lines engineered for tetracycline‐regulated expression of tumor suppressor candidate genes from a frequently affected chromosomal region, 3p21

We modified a tetracycline‐regulated system that can control the activity of individual genes quantitatively and reversibly in transgenic mammals. Despite these advances, there remained one problem in the intensive use of the tet‐system: the limited range of acceptor cell lines, expressing a tetracycline‐controlled transcriptional activator (tTA). This study describes in detail new vectors and a unifying strategy to generate tTA‐expressing cell lines.

Tryptophan Hydroxylase-1 Gene Variants Associate with a Group of Suicidal Borderline Women

Alterations in the serotonin (5-HT) system have been related to impulsive aggression and suicidal behavior, common features of the borderline personality disorder (BPD). Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in 5-HT biosynthesis. Two isoforms are known, TPH-1 and TPH-2. TPH-1 has been correlated to various psychiatric and behavioral disorders by gene polymorphism association studies. We aimed to determine whether specific TPH-1 haplotypes associate with BPD. A case–control design was employed. The control group included 98 women without psychiatric history. In all, 95 patients were included, all Caucasian women with a BPD diagnosis who had attempted suicide at least twice during their lifetime. Exclusion criteria were: (i) substance dependence; (ii) dementia or other irreversible organic brain syndromes; (iii) psychotic disorders or major depressive illness with melancholic features; (iv) life-threatening eating disorders. Six single-nucleotide polymorphisms (SNPs) were found at significant linkage disequilibrium across 23 kb of the TPH-1 gene in both patients and controls, suggesting a haplotype block structure. While no individual SNP showed association, several haplotypes associated with the BPD group. In particular, one six-SNP haplotype was absent from the control group while representing about one-quarter of all haplotypes in the BPD group (corrected P≪10−5). A ‘sliding window’ analysis attributed the strongest disease association to haplotype configurations located between the gene promoter and intron 3. We conclude that TPH-1 associates with BPD in suicidal women. Our data support the expectation that haplotype analysis is superior to single locus analysis in gene–disease, case–control association studies.

DNA-Encoding Enzymatically Active HIV-1 Reverse Transcriptase, but Not the Inactive Mutant, Confers Resistance to Experimental HIV-1 Challenge

The present study was undertaken to examine the immunogenicity of a single plasmid DNA representing the reverse transcriptase (RT) of HIV-1. Plasmids containing the enzymatically active RT as well as a mutated nonenzymatically active RT with nucleotide (nt)-binding motifs of YMDD and YMLL, respectively, were used to immunize mice. Both constructs induced similar good antibody and T cell responses, with a tendency towards antibody directed to peptides representing the active and mutated sites. Immunized mice were challenged with a murine pseudotype HIV-1/MuLV infected spleen cells. Seven out of 10 mice immunized with RT had no recoverable HIV-1, while 10 individuals immunized with the RT mutant and all the 18 controls had high levels of recoverable HIV-1. This indicates that mutation of RT reduces the desired immunogenicity.

Human chromosome 3: high-resolution fluorescencein situ hybridization mapping of 40 uniqueNotl linking clones homologous to genes and cDNAs

Forty newNotl linking clones representing sequence tagged sites (STSs) were mapped by fluorescencein 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 γ-subunit gene and was mapped to 3q23–q24. To our knowledge, this is the first time this gene has been mapped. OneNoti linking clone (NL1-229) probably contains a new protein phosphatase gene. This clone was mapped to 3p25. FiveNoti 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.

Increase of Faecal Tryptic Activity Relates to Changes in the Intestinal Microbiome: Analysis of Crohn's Disease with a Multidisciplinary Platform

Objective To investigate—by molecular, classical and functional methods—the microbiota in biopsies and faeces from patients with active Crohns disease (CD) and controls. Design The microbiota in biopsies was investigated utilizing a novel molecular method and classical cultivation technology. Faecal samples were investigated by classical technology and four functional methods, reflecting alterations in short chain fatty acids pattern, conversion of cholesterol and bilirubin and inactivation of trypsin. Results By molecular methods we found more than 92% similarity in the microbiota on the biopsies from the two groups. However, 4.6% of microbes found in controls were lacking in CD patients. Furthermore, NotI representation libraries demonstrate two different clusters representing CD patients and controls, respectively. Utilizing conventional technology, Bacteroides (alt. Parabacteroides) was less frequently detected in the biopsies from CD patients than from controls. A similar reduction in the number of Bacteroides was found in faecal samples. Bacteroides is the only group of bacteria known to be able to inactivate pancreatic trypsin. Faecal tryptic activity was high in CD patients, and inversely correlated to the levels of Bacteroides. Conclusions CD patients have compositional and functional alterations in their intestinal microbiota, in line with the global description hypothesis rather than the candidate microorganism theory. The most striking functional difference was high amount of faecal tryptic activity in CD patients, inversely correlated to the levels of Bacteroides in faeces.

hUNC93B1: a novel human gene representing a new gene family and encoding an unc-93-like protein.

We have identified a novel human gene UNC93B1 encoding a protein related to unc-93 of Caenorhabditis elegans. The combined sequence derived from several cDNA clones is 2282 bp and comparison with genomic sequence shows that the gene contains 11 exons. The longest open reading frame encodes a deduced sequence of 597 amino acids. Homology analysis shows that the hUNC93B1 gene is highly conserved and related to sequences in Arabidopsis thaliana, C. elegans, Drosophila melanogaster, chicken and mouse. Structural analysis of the deduced amino acid sequence of hUNC93B1 points to possible existence of multiple membrane-spanning domains. hUNC93B1 protein also displays some similarities to the bacterial ABC-2 type transporter signature and to ion transporters of Deinococcus radiodurans and Helicobacter pylori. As revealed by Northern analysis, the level of expression varies significantly between tissues, with the highest level detected in the heart. The gene was mapped to chromosomal band 11q13 by fluorescence in situ hybridization. We suggest that this gene is a member of a novel hUNC93B-related gene family.

Haplotype analysis confirms association of the serotonin transporter (5-HTT) gene with schizophrenia but not with major depression†

Serotonin (5‐HT) has been implicated in the pathophysiology of several psychiatric disorders including major depressive disorder (MDD) and schizophrenia (SCZ). The serotonin transporter (5‐HTT) is a major regulator of 5‐HT function. 5‐HTT gene polymorphic variants have been associated with both MDD and SCZ. A case–control design was used for candidate gene‐disease association in 194 MDD patients, 155 schizophrenic psychosis patients, and 246 healthy controls, all North European Caucasians. Four polymorphisms were analyzed in terms of genotype, allele, and haplotype‐based associations. Linkage disequilibrium (LD) analysis was also carried out. Bonferroni correction was used for multiple testing. Haplotype‐based analyses showed significant associations between 5‐HTT and SCZ but not MDD. No single locus associations were observed. In agreement with published meta‐analysis our results indicate that 5‐HTT associates with SCZ but not with MDD. It appears that risk for SCZ maps within a specific 5‐HTT haplotype block.

Assignment of the GPR14 gene coding for the G-protein-coupled receptor 14 to human chromosome 17q25.3 by fluorescent in situ hybridization.

The orphan G-protein-coupled receptors (GPRs) constitute an abundant family of membrane receptors of high pharmacological interest (Wilson et al., 1998). The NCBI BLASTX analysis revealed that partial sequence of our NotI linking clone NB1-680 isolated from a human NotI linking library (Zabarovsky et al., 1994) displays 78% identity with rat Gpr14 (Marchese et al., 1995) in a 123 aa overlap. The sequenced region was extended to 1714 bp. This sequence reveals a 389 aa open reading frame with high similarity to GPRs. Recently a new member of this family, human GPR14, has been cloned (Ames et al., 1999). This gene is expressed mainly in cardiovascular tissue and the GPR14 protein is able to mobilize intracellular Ca2+ in response to human urotensin-II and effectively constricts arteries. The translated sequence of NB1-680 is 100% identical to GPR14 protein in a 389 aa overlap (or 99% over a 1,166-bp overlap, two nucleotide changes). The only amino acid difference is Asp 235 in our protein instead of Ala 235 in GPR14. Most probably this difference reflects polymorphism and thus it means that our NB1-680 contains the GPR14 gene.

Mutations Conferring Drug Resistance Affect Eukaryotic Expression of HIV Type 1 Reverse Transcriptase

Mutations in reverse transcriptase (RT) confer high levels of HIV resistance to drugs. However, while conferring drug resistance, they can lower viral replication capacity (fitness). The molecular mechanisms behind remain largely unknown. The aim of the study was to characterize the effect of drug-resistance mutations on HIV RT expression. Genes encoding AZT-resistant RTs with single or combined mutations D67N, K70R, T215F, and K219Q, and RTs derived from drug-resistant HIV-1 strains were designed and expressed in a variety of eukaryotic cells. Expression in transiently transfected cells was assessed by Western blotting and immunofluorescent staining with RT-specific antibodies. To compare the levels of expression, mutated RT genes were microinjected into the nucleus of the oocytes of Xenopus laevis. Expression of RT was quantified by sandwich ELISA. Relative stability of RTs was assessed by pulse-chase experiments. Xenopus oocytes microinjected with the genes expressed 2-50 pg of RT mutants per cell. The level of RT expression decreased with accumulation of drug-resistance mutations. Pulse-chase experiments demonstrated that poor expression of DR-RTs was due to proteolytic instability. Instability could be attributed to additional cleavage sites predicted to appear in the vicinity of resistance mutations. Accumulation of drug-resistance mutations appears to affect the level of eukaryotic expression of HIV-1 RT by inducing proteolytic instability. Low RT levels might be one of the determinants of impaired replication fitness of drug-resistant HIV-1 strains.