RECODE 2003

The RECODE database is a compilation of translational recoding events (programmed ribosomal frameshifting, codon redefinition and translational bypass). The database provides information about the genes utilizing these events for their expression, recoding sites, stimulatory sequences and other relevant information. The Database is freely available at http://recode.genetics.utah.edu/.     

Molecular genetic analysis of Y-chromosome micro deletions in men with severe spermatogenic defects

Microdeletions of the Y-chromosomal AZF loci were revealed in 10 (12%) of 82 patients with severe idiopathic spermatogenetic defects. Deletions involved AZFc in six patients, AZFa in one patient, AZFb + c in two patients, and AZFa + b + c in one patient. Microdeletion analysis employed multiplex PCR with 22 pairs of primers directed to Y-specific STS of deletion intervals 5, 6, and 7 (Yq11). Spermatogenesis in men with AZF microdeletions was assessed with semen analysis, microscopic examination of testicular aspirate, and quantitative karyotypic analysis of immature germline cells in ejaculate or aspirate. The character of spermatogenetic defects was correlated with the size and location of microdeletions in order to study the genotype-phenotype relationship.     

The Mutation Spectrum of the CFTR Gene in Cystic Fibrosis Patients from Bashkortostan

Mutations of CFTR were studied in patients with cystic fibrosis (CF) from Bashkortostan. In total, 15 mutations were observed and 51% of all mutant alleles identified. The most diagnostically significant mutations were delF508 (33.8%), 394delTT (3.52%), CFTRdele2,3(21kb) (1.41%), R334W (1.41%), 3849 + 10kbC T (1.41%), and N1303K (1.41%). Mutations G542X, 2184insA, S1196X, and W1282X were each found in less than 1% patients. Five new mutations and two neutral substitutions were revealed. These were I488M (exon 10), 1811 + 12A C (intron 11), T663S (exon 13), I1226R (exon 19), 4005 + 9A C (intron 20), 2097A C (A655A, exon 13), and 3996G C (V1288V, exon 20). Bashkortostan was shown to differ in the CFTR mutation spectrum from other regions of Russia. The results will allow direct DNA diagnostics of CF in far more families. Molecular screening of probands" relatives will contribute to identification and medical genetic counseling of heterozygous carriers, which is essential for CF prevention.     

Reduction of 1-methyl-1-nitrosourea-induced tumor burden with DNA vaccines encoding mutated ras epitopes and the costimulatory molecule B7.1

1-methyl-1-nitrosourea (MNU), a well characterized carcinogen, was used to induce adenocarcinomas in rat mammary gland. 150 days after the first injection of MNU, the animals were treated with DNA minigene vaccines encoding ras T cell epitopes together with the co-stimulatory molecule B7.1 (CD 80). Five injections with a biolistic device (gene gun) in monthly intervals significantly reduced the tumor burden. A therapeutic effect could be measured with both, DNA vaccines encoding ras epitopes and B7.1, as well as with a DNA vaccine expressing solely the B7.1 molecule thus indicating the potential of genetic vaccination for turnor treatment.     

Translational recoding signals between gag and pol in diverse LTR retrotransposons

Because of their compact genomes, retroelements (including retrotransposons and retroviruses) employ a variety of translational recoding mechanisms to express Gag and Pol. To assess the diversity of recoding strategies, we surveyed gag/pol gene organization among retroelements from diverse host species, including elements exhaustively recovered from the genome sequences of Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Candida albicans, and Arabidopsis thaliana. In contrast to the retroviruses, which typically encode pol in the -1 frame relative to gag, nearly half of the retroelements surveyed encode a single gag-pol open reading frame. This was particularly true for the Ty1/copia group retroelements. Most animal Ty3/gypsy retroelements, on the other hand, encode gag and pol in separate reading frames, and likely express Pol through +1 or -1 frameshifting. Conserved sequences conforming to slippery sites that specify viral ribosomal frameshifting were identified among retroelements with pol in the -1 frame. None of the plant retroelements encoded pol in the -1 frame relative to gag; however, two closely related plant Ty3/gypsy elements encode pol in the +1 frame. Interestingly, a group of plant Ty1/copia retroelements encode pol either in a +1 frame relative to gag or in two nonoverlapping reading frames. These retroelements have a conserved stem-loop at the end of gag, and likely express pol either by a novel means of internal ribosomal entry or by a bypass mechanism.     

The BCL-xL and ACR-1 Genes Promote Differentiation and Reduce Apoptosis in Muscle Fibers of mdx Mice

The effects of the human BCL-xL and ACR-1genes on dystrophin expression in cross-striated muscle fibers (CSMF) and on CSMF viability were studied in mdx mice after ballistic cotransfection with the human dystrophin minigene. In control mice, the proportion of dystrophin-positive (D(+)) and dying CSMF were 2.1 ± 0.1 and 2.1 ± 0.3%, respectively. Introduction of the dystrophin minigene (20 g of the pSG5dys plasmid) increased the proportions of D(+) and dying CSMF to 5.6 ± 1.4% and 4.5 ± 0.9%, respectively. When pSG5dys was introduced along with the pSFFV-Neo plasmid carrying the BCL-xL gene (10 g of each plasmid per shot), the death of CSMF decreased to 3.7 ± 1% and the proportion of D(+) CSMF significantly (P < 0.05) increased to 12.2 ± 2.2%. Cotransfection with the dystrophin minigene and the BCL-xL gene at 20 g of each plasmid per shot did not stimulate generation of D(+) CSMF, but did reduce the CSMF death to 1.5 ± 0.3%. Introduction of pSG5dys along with the pRc-CMV-10.1 plasmid containing the ACR-1 gene (10 g of each plasmid per shot) reduced the proportion of D(+) CSMF to 1.1 ± 0.5% and significantly reduced the proportion of dying CSMF to 0.9 ± 0.3% as compared with the proportions observed in intact mice or in mice subjected to transfection with pSG5dys. Introduction of the pSG5dys plasmid substantially reduced the proportion of CSMF with peripheral nuclei, suggesting disturbed CSMF differentiation. After cotransfection with the human dystrophin minigene, the BCL-xL and ACR-1 genes did not affect the extent of CSMF differentiation as compared with that observed in the case of the dystrophin minigene alone. Thus, ballistic transfection of mdx mice with the human dystrophin gene used along with the BCL-xLor ACR-1 gene was shown to suppress the death of muscle fibers and to expedite dystrophin synthesis and cell differentiation.     

Analysis of association of Col1a1 gene alleles with the development of osteoporosis

Allele frequencies of the G-->T polymorphism at the regulatory region of the Collal gene in the population of the northwestern Russia (control group) and in osteoporotic patients were estimated by the RFLP method based on PCR-mediated site-directed mutagenesis. Three patient groups with radiologically confirmed osteoporosis were examined. Group 1 consisted of 64 patients with severe osteoporosis complicated by fractures (SO); group 2 included 15 children with idiopathic osteoporosis (IO); group 3 consisted of 98 women with postmenopausal osteoporosis developed at the background of estradiol-deficiency state (PMO). The frequency of functionally defective allele s in the control group was 16.7%. It was statistically different from that in the SO patients (48.4%) (P < 0.01) and in the IO children (40%) (P < 0.01). The frequency of allele s in the PMO patients constituted 23.0% and it was similar to that in the control group (P > 0.05). Analysis of the Collal alleles provides early detection of the individuals with hereditary predisposition to osteoporosis and prophylaxis of the disease at the presymptomatic stage.     

PCR assay of DNA damage and repair at the gene level in brain and spleen of gamma-irradiated young and old rats.

The PCR amplification of fragments of transcribed (beta-actin, p53) and nontranscribed (IgE, heavy chain) genes in brain and spleen DNA from gamma-irradiated and unirradiated 2- and 28-month-old rats was studied. The amplification levels of fragments of these genes in DNA from old rats were substantially lower than those from young rats, which suggested that these gene fragments in old-rat DNA contained lesions blocking thermostable polymerase in PCR. The beta-actin and IgE gene fragments of spleen DNA from old rats exhibited a significantly higher level of lesions inhibiting Tth polymerase compared to analogous fragments of brain DNA from the same animals. DNA from the tissues of gamma-irradiated rats showed the amount of damage inhibiting amplification to be dependent on animal age and the postirradiation time before DNA isolation. As judged from the changes in the amplification level of gene fragments, there was no preferential fast repair of lesions in the actively transcribed gene beta-actin compared to the nontranscribed gene IgE (heavy chain) in the brain and spleen of gamma-irradiated young and old rats. The amplification results suggest that equal amounts of DNA lesions were repaired in the brain of both old and young rats during the first 0.5 h of the postirradiation time (fast-repair phase), whereas in the subsequent postirradiation period over 5 h (slow-repair phase), the efficiency of damage elimination in the brain DNA of old rats was markedly lower. As for the spleen tissue, the elimination of lesions blocking Tth polymerase was much lower in old gamma-irradiated animals for both of the repair phases.     

Cytogenetic analysis of human spermatozoa using intracytoplasmic sperm injection into mouse oocytes

The chromosome complement of human spermatozoa has been analyzed after their intracytoplasmic injection into unfertilized mouse oocytes. A total of 427 metaphase plates have been obtained, including 176 metaphase plates from spermatozoa with normal head morphology (108 and 68 spermatozoa from patients with normal (the control group) and abnormal spermogram parameters, respectively), and 251 metaphase plates from spermatozoa with abnormal heads (76, 91, 67, and 17 spermatozoa with large, amorphous, elongated, and round heads, respectively). The frequency of chromosome abnormalities in the control group is 26.1%, with hyperploidy, hypoploidy, and structural aberrations accounting for 7.4, 12.3, and 6.4% of the abnormalities, respectively. In none of the groups did the ratio between the numbers of X- and Y-bearing spermatozoa significantly differ from 1 : 1. The diploidy frequency was significantly higher in spermatozoa with large and amorphous heads compared to the control group (2.36, 3.29, and 0%, respectively). None of the groups of spermatozoa differed from the control group with respect to the frequency of structural aberrations. The type of the abnormal head morphology has been found to be correlated with the sperm chromosome complement.