A Matrix Associated Region Localizes the Human SOCS-1 Gene to Chromosome 16p13.13

The MarFinder algorithm was applied to a newly sequenced segment of 16p13.13 abutting the 3 end of the human PRM1 PRM2 TNP2 locus. A candidate region of matrix attached was identified. Subsequent biophysical analysis showed that this region was attached to the somatic nuclear matrix. Nucleotide sequence analysis also revealed the presence of a CpG island. Data base queries showed that this region contained the SOCS-1 gene. Thus, the SOCS-1 gene is bounded by a somatic MAR and is just 3 of the spermatid-expressed PRM1 PRM2 TNP2 domain at position 16p13.13.     

Polymerase chain reaction-single strand conformation polymorphism analysis of the p53 gene in paraffin-embedded surgical material from human renal cell carcinomas

p53 tumour suppressor gene mutations were studied in 118 renal cell carcinomas using paraffin-embedded surgical material. Optimal results were obtained with analysis of exon lengths between 150 and 200 base pairs for polymerase chain reaction. Single strand conformation polymorphism and sequencing analysis revealed only two point mutations (2/118, 2%): one involving codon 135; TGCTTC (cysteinephenylalanine) and the other codon 175; CGCCAC (argininehistidine). Both of these cases were classified as granular cell subtype on microscopic observation. The data suggest that the p53 tumour suppressor gene is not related to tumour initiation, promotion, or progression of renal cell carcinomas. However, there is the possibility that granular cell type carcinomas may have a different genetic background from clear cell type renal neoplasms.     

Altered expression of the steroid bioconverting pathway in pAN 7-1 transformants of Cochliobolus lunatus

Summary The filamentous fungus C. lunatus converts progesterone mainly to its 11-hydroxy derivative. C. lunatus transformed with the plasmid pAN 7-1, which contains the E. coli hph gene expressed under the control of the A. nidulans gpd and trpC expression signals, lacks this activity, but exhibits acetyl side chain degradation of progesterone through the reaction scheme progesterone20-hydroxy-progesterone ⁴-androstene-3,17-dione testolactone+testosterone. The main partof this metabolic pathway is not expressed in the non-transformed strain. It was determined that the site-specific integration of the plasmid into the genome directly influences the expression of genes involved in the bioconversion of steroids.     

Distribution of C→T and T→C polymorphisms of the urokinase-type plasminogen activator gene in children with type 1 diabetes mellitus and insulin resistance

Abstract We analysed the distribution of genotypes and frequency of alleles of two polymorphisms in the urokinase-type plasminogen activator (uPA) gene: a CT substitution in exon 6 and a TC substitution in intron 7 in 89 children with type 1 diabetes mellitus and insulin resistance compared with 120 non-diabetic control subjects. All genotypes were determined by the allele-specific polymerase chain reaction. We found that the frequency of the T/T homozygote (15%) in the patient group was significantly (P<0.05) higher than in the controls (7%). There were no differences in the distribution of the TC polymorphism between patients and controls, which suggests that this genetic change is probably phenotypically silent. In conclusion, our results indicate that the higher percentage of T/T homozygotes in patients might be associated with T1DM coexisting with insulin resistance.     

Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae

We have studied the influence of a temperature-sensitive cdc2-1 mutation in DNA polymerase on the selection-induced mutation occurring at the LYS-2 locus in the yeast Saccharomyces cerevisiae. It was found that in cells plated on synthetic complete medium lacking only lysine, the numbers of Lys⁺ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. When cdc2-1 mutant cells, after selective plating, were incubated at the restrictive temperature of 37°C for 5 h daily for 7 days, the frequency of an adaptive reversion of lys ^- Lys⁺ was significantly higher than the frequency in cells incubated only at the permissive temperature, or in wild-type cells incubated either at 23°C or 37°C. Therefore, when the proof-reading activity of DNA polymerase is impaired under restrictive conditions, the frequency of adaptive mutations is markedly enhanced.     

Activation of precursors of T killers of hematopoietic stem cells by thymosine

Lymph node cells from normal CBA mice, from CBACBA syngeneic radiation chimeras, and B mice were incubatedin vitro with fraction 5 of thymosine, and transplanted into sublethally irradiated (CBA x C57BL)F₁ recipients, and the number of endogenous colonies in the recipients' spleen was determined. Thymosine was shown to potentiate the killer activity of lymph node cells of normal CBA mice and of CBACBA syngeneic radiation chimeras, but not of B mice. It is suggested that the target for the action of thymosine is the subpopulation of T₁ lymphocytes.Academician of the Academy of Sciences of the USSR.Institute of Biophysics, Ministry of Health of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 5, pp. 602–605, May, 1978.     

Human gene for proliferating cell nuclear antigen has pseudogenes and localizes to chromosome 20

We have isolated from a human genomic library a pseudogene of the proliferating cell nuclear antigen (PCNA)gene. Its sequence shows a 78% similarity with the human PCNA/cDNA. The PCNAgene is located on human chromosome 20, while the pseudogene maps to chromosome region XpterXq13. An additional locus detected by the full-length PCNA cDNA, but not by intron probes, segregates concordantly with chromosome region 6p126pter and probably represents a second pseudogene.     

Mapping of the genes for human endoplasmic reticular heat shock protein gp96/grp94

The murine tumor rejection antigen gp96 (TRA1, mapped to mouse chromosome 10) is a member of the heat shock protein family. Using a fragment of the murine gp96 cDNA as a probe, three gp96-related human genes have been isolated and structurally characterized. They have been mapped to human chromosomes 1 (p22), 12 (q24.2 q24.3), and 15 (q25 q26) by Southern blot hybridization and in situ hybridization of gene-specific probes. Only one of the genes, designatedTRA1 (human chromosome 12) is a coding gene; the other genes (TRA1P1 andTRAP2) appear to be independently derived, processed pseudogenes.     

Identification of a novel 2026G→C mutation of the MRP2 gene in a Japanese patient with Dubin-Johnson syndrome

Dubin-Johnson syndrome is a recessive inherited disorder with conjugated hyperbilirubinemia caused by a dysfunction of multidrug resistance protein 2 (MRP2) on the canalicular membrane of hepatocytes. A mutational analysis of the MRP2 gene was carried out in three Japanese patients and their family members. In two patients, the homozygous mutations c.1901del67 and c,2272del168 were found. In the third patient, a –24CT polymorphism and the two mutations c.1901del67 and 2026GC were detected. The 2026GC mutation was a novel mutation in exon 16 affecting the conversion of Gly⁶⁷⁶ to Arg⁶⁷⁶ (G676R) in the MRP2 protein, and was not detected in fifty healthy volunteers. The G676R mutation was located in the Walker A motif of the first nucleotide binding domain in the MRP2 protein, and it was suggested that the mutation induced the dysfunction of the MRP2 protein. It was concluded that the compound heterozygosity of the two mutations of the MRP2 gene in the third patient contributed to the induction of hyperbilirubinemia in this case.     

Protein secretion in yeast: Two chromosomal mutants that oversecrete killer toxin in Saccharomyces cerevisiae

Summary Two chromosomal mutations in yeast that result in oversecretion of the K₁ killer toxin protein were examined. A recessive mutation in gene ski5 appears to lead to toxin oversecretion through a defect in a cell surface, PMSF-inhibited protease. A wild type killer strain degraded toxin following synthesis, and degradation could be partially prevented by addition of PMSF to the growth medium. The ski5 mutation caused an approximate ten fold oversecretion of toxin, similar to that seen in a PMSF-treated wild type culture, and no increased oversecretion in the presence of PMSF. The ski5 mutation caused oversecretion of other low molecular weight secreted proteins and appeared to oversecrete the -factor pheromone, as judged by activity tests. The ski5 mutation was complemented by mutations in ski genes 1–4, and the mutant was not supersensitive to mating pheromones or K₂ killer toxin.We also examined killer strains with a mutation in the nuclear gene krel which results in a defective (16)--D-glucan cell wall receptor for killer toxin. Such strains oversecrete toxin into the growth medium, but also, unexpectedly, oversecrete most other secreted proteins. The defect in (16)--D-glucan in these mutants appears to perturb the partitioning of secreted proteins between the cell wall and the medium.     

Human interstitial retinol-binding protein (IRBP): cloning,partial sequence,and chromosomal localization

A cloned 2184-bp cDNA coding for human interstitial retinol-binding protein (IRBP) has been isolated and sequenced. The probe hybridized to a 5.2-kb poly(A) RNA from human retinas. Nineteen tryptic peptides (363 amino acids) sequenced and purified from bovine IRBP could be aligned with 86–88% homology to the translated sequence. Two segments approximately 200 amino acids long were found to have a 41% residue identity,suggesting an internal duplication event. This cloned cDNA was used to probe DNA samples from a panel of 29 rodent-human somatic cell hybrids, mapping the structural gene for IRBP to chromosome 10. In situ hybridization suggested a regional localization near the centromere (p11.2q11.2), although a secondary site of hybridization at q2425 was also observed.     

Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris)

Summary Variation within the internal transcribed spacers (ITS1 and ITS2) and 5.8s ribosomal DNA gene of the heterothallic phytopathogenic filamentous fungus, Fusarium sambucinum (teleomorph=Gibberella pulicaris), was examined in 86 strains from diverse geographical locations by PCR amplification and direct sequencing in order to measure intraspecific divergence within the ITS region. Sequence analysis revealed three ITS types (A, B, C), within which divergence was extremely low (0–2.3%). Surprisingly, the level of intraspecific divergence observed between ITS types, AB=14.3%, AC=15%, and BC=4.6%, is much greater than that reported for any other species. The degree to which transition/transversions and insertion/deletions make up the pattern of ITS sequence evolution both within and between types was analyzed. The sequences of the ITS types exhibit a C-T transition bias together with a GC insertion/deletion bias. In comparison, the genic flanking sequences, including the 5.8s rDNA gene and 5 end of the 28s large nuclear rDNA, are highly conserved. By the criteria of mating and DNA-DNA hybridization, all the strains examined represent a single species. Discordance between the ITS sequence data and other molecular and genetic data on F. sambucinum is discussed.     

Mapping translocation breakpoints by orthogonal field agarose-gel electrophoresis

Orthogonal field agarose-gel electrophoresis (OFAGE) of chromosomes from translocation-bearing and normalNeurospora crassa strains was utilized, first, to recover cosmids from a translocated region, and second, to map translocation breakpoints. Surprisingly, the right breakpoints in two independently derived, interstitial translocations,T(II III) AR18 andT(II VI)P2869, are within about 5.6 kbp of each other suggesting that this region of linkage group (LG) II may be fragile or otherwise subject to chromosome breakage. Mapping translocation breakpoints through OFAGE, or other similar methods, should allow for DNA sequencing across breakpoints that are not associated with mutant phenotypes or that are not within walking distance of cloned markers.     

Association of eotaxin-2 gene polymorphisms with plasma eotaxin-2 concentration

Chemokine (C–C motif) ligand 24 (CCL24, eotaxin-2) is a CC chemokine that recruits and activates cells bearing the CC chemokine receptor 3, which play a major role in asthma. Previously, we observed a significant association between a single nucleotide polymorphism (SNP) in eotaxin-2 (CCL24+1272A G) and a lower risk of asthma. Consequently, this study has followed up on those genetic effects by evaluating the association between the SNP and plasma eotaxin-2 concentration in 172 asthmatics and 135 normal controls. Asthmatics had significantly higher plasma eotaxin-2 levels than did normal controls (P=0.02). The SNP (CCL24+1272A G) and two haplotypes (ht2 and ht6) were strongly associated with plasma eotaxin-2 levels in asthmatics (CCL24+1272A G: P=0.006, ht2: P=0.006, and ht6: P=0.002). The CCL24+1272A G allele and the ht2 and ht6 haplotypes showed a gene–dose effect on the plasma eotaxin-2 levels in asthmatics (P=0.005–0.032). In conclusion, the susceptibility of patients with asthma to high eotaxin-2 production may be due to genetic effects of the CCL24+1272A G polymorphism, ht2 and ht6 haplotypes.     

Tyrosinaemia type I—de novo mutation in liver tissue suppressing an inborn splicing defect

Many patients with tyrosinaemia type 1 have a mosaic pattern of fumarylacetoacetase (FAH) immunopositive or immunonegative nodules in liver tissue. This phenomenon has been explained by a spontaneous reversion of the mutation in one allele to a normal genotype, but only a few nodules have been examined. We now report on a Norwegian patient, compound heterozygous for the mutations IVS12g⁺⁵a and G¹⁰⁰⁹A, with liver mosaicism, but with an immunopositive nodule in which both primary mutations were intact. In the immunopositive hepatocytes of this nodule, genetic analyses showed a new mutation, C¹⁰⁶¹A, 6 bp upstream of the primary mutation IVS12g⁺⁵a in the FAH gene. The splicing defect caused by the primary mutation is most likely suppressed by the new mutation due to improvement of the splicing site. In the same liver we demonstrate another nodule of regenerating immunopositive tissue due to reversion of one of the primary mutations to a normal genotype. Together with the original cells this makes a triple mosaicism of hepatocytes with one, two or three point mutations in the FAH gene.     

The chloroplast gene cluster containing psbF,psbL, petG and rps3 is conserved in Chlamydomonas

We have sequenced a 6.8-kb segment of the Chlamydomonas eugametos chloroplast DNA which contains the psbF, psbL, petG and rps3 genes. As in the distantly related green alga Chlamydomonas reinhardtii, these genes reside in this order (53) on the same DNA strand, suggesting that such a chloroplast gene cluster was present in the most recent common ancestor of all Chlamydomonas species. For each of the four genes, with the exception of rps3, the C. eugametos and C. reinhardtii coding regions were found to be identical, or very similar, in length, whereas each of the intergenic spacers is substantially longer in C. eugametos than in C. reinhardtii. The central portion of both Chlamydomonas rps3 genes features a long extra coding region relative to other rps3 sequences. We have shown that the insertion sequence in the C. eugametos rps3 is not excised at the RNA level.     

Epitope-tagging vectors designed for yeast

In order to facilitate the process of epitope-tagging of yeast proteins, we have constructed two Saccharomyces cerevisiae-Escherichia coli shuttle vectors that allow fusion of a sequence encoding an epitope of the human c-myc protein at the 3 end of any gene. An example of the use of this technique is presented.     

Association of polymorphisms of paraoxonase 1 and 2 genes,alone or in combination,with bone mineral density in community-dwelling Japanese

Oxidative stress may affect cellular functions in various pathological conditions, including osteoporosis. Paraoxonase 1 confers antioxidant properties on high-density lipoprotein, with which it is associated, by reducing the accumulation of lipid peroxidation products. We have now examined whether the 584AG (Gln192Arg) and 172TA (Leu55Met) polymorphisms of the paraoxonase 1 gene and the 959GC (Cys311Ser) polymorphism of the paraoxonase 2 gene are associated with bone mineral density (BMD) in community-dwelling Japanese (1,087–1,094 women and 1,112–1,125 men). The subjects were aged 40 –79 years and were randomly recruited to a population-based prospective cohort study of aging and age-related diseases. BMD for the lumbar spine and right femoral neck was measured by dual-energy X-ray absorptiometry. Genotypes were determined with a fluorescence- or colorimetry-based allele-specific DNA primer-probe assay system. The 584AG and 172TA polymorphisms of the paraoxonase 1 gene and the 959GC polymorphism of the paraoxonase 2 gene were associated with BMD for the lumbar spine or femoral neck in postmenopausal women, with the 584GG, 172TT, and 959CC genotypes representing risk factors for reduced bone mass. None of these three polymorphisms was associated with BMD in premenopausal women or in men. Our results suggest that the paraoxonase 1 and 2 genes are candidate loci for reduced bone mass in postmenopausal Japanese women.     

Specific changes in the M1 protein during adaptation of influenza virus to mouse

Summary The matrix (M) gene of influenza virus has been implicated as a determinant of virulence for mouse brain and lung. Comparison of the M gene sequences of the mouse brain adapted variants A/NWS/33 and A/WSN/33 to their parent, A/WS/33, identified two specific amino acid substitutions in the M₁ protein which correlated with virulence for mouse: Ala⁴¹ Val and Thr¹³⁹ Ala.