cDNA cloning of a novel heterogeneous nuclear ribonucleoprotein gene homologue in Caenorhabditis elegans using hamster prion protein cDNA as a hybridization probe.

The mammalian prion protein (PrPc) is a cellular protein of unknown function, an altered isoform of which (PrPsc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. The evolutionary conservation of the PrP gene has been reported in the genomes of many vertebrates as well as certain invertebrates. In the genome of nematode Caenorhabditis elegans, the sequence capable of hybridizing with the mammalian PrP cDNA probe has been demonstrated, predicting the presence of the PrP gene homologue in C.elegans. In this study, Southern analysis with the hamster PrP cDNA (HaPrP) probe confirmed the previous observation. Moreover, Northern analysis revealed that the sequence is actively transcribed in adult worms. Thus, we screened C.elegans cDNA libraries with the HaPrP probe and isolated a cDNA that hybridizes to the same sequence in C.elegans that hybridized with the HaPrP probe in the Southern and Northern analyses. The deduced amino acid sequence of this cDNA, however, is substantially homologous with heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins rather than mammalian PrPc. The hnRNPs contain the glycine-rich domain in the C-terminal half of the molecule, which also seemed to be in PrPc at the N-terminal half of the molecule. Both of the glycine-rich domains are composed of tracts with high G + C content, indicating that these tracts may due to the hybridizing signals. These results suggest that this cDNA clone is derived from a novel hnRNP gene homologue in C.elegans but not from a predicted PrP gene homologue.     

Activity of chimeric RNAs of U6 snRNA and (-)sTRSV in the cleavage of a substrate RNA.

U6 small nuclear RNA is one of the spliceosomal RNAs essential for pre-mRNA splicing. Discovery of mRNA-type introns in the highly conserved region of the U6 snRNA genes led to the hypothesis that U6 snRNA functions as a catalytic element during pre-mRNA splicing. The highly conserved region of U6 snRNA has a structural similarity with the catalytic domain of the negative strand of the satellite RNA of tobacco ring spot virus [(-)sTRSV], suggesting that the highly conserved region of U6 snRNA forms the catalytic center. We examined whether synthetic RNAs consisting of the sequence of the highly conserved region of U6 snRNA or various chimeric RNAs between the U6 region and the catalytic RNA of (-)sTRSV could cleave a substrate RNA that can partially base-pair with them and have a GU sequence. Chimeric RNAs with 70 to 83% sequence identity with the conserved region of S. pombe U6 snRNA cleaved the substrate RNA at the 5' side of the GU sequence, which is shared by the 5' end of an intron in a pre-mRNA. We found that the highly conserved region of U6 snRNA and the catalytic domain of (-)sTRSV are strikingly similar in structure to the catalytic core region of the group I self-splicing intron in cyanobacteria. These results suggest that U6 snRNA, (-)sTRSV and the group I self-splicing intron originated from a common ancestral RNA, and support the hypothesis that U6 snRNA catalyzes pre-mRNA splicing reaction.     

A pyrimidine-guanine sequence-specific ribonuclease from Rana catesbeiana (bullfrog) oocytes.

A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS.     

Ataxia-telangiectasia: Linkage analysis in highly inbred Arab and Druze families and differentiation from an ataxia-microcephaly-cataract syndrome

Summary Ataxia-telangiectasia (A-T) is a progressive autosomal recessive disease featuring neurodegeneration, immunodeficiency, chromosomal instability, radiation sensitivity and a highly increased proneness to cancer. A-T is ethnically widespread and genetically heterogeneous, as indicated by the existence of four complementation groups in this disease. Several A-T-like genetic diseases share various clinical and cellular characteristics with A-T. By using linkage analysis to study North American and Turkish A-O families, the ATA (A-T, complementation group A) gene has been mapped to chromosome 11q23. A number of Israeli Arab A-T patients coming from large, highly inbred families were assigned to group A In one of these families, an additional autosomal recessive disease was identified, characterized by ataxia, hypotonia, microcephaly and bilateral congenital cataracts. In two patients with this syndrome, normal levels of serum immunoglobulins and alpha-fetoprotein, chromosomal stability in peripheral blood lymphocytes and skin fibroblasts, and normal cellular response to treatments with X-rays and the radiomimetic drug neocarzinostatin indicated that this disease does not share, with A-T, any additional features other than ataxia. These tests also showed that another patient in this family, who is also mentally retarded, is affected with both disorders. This conclusion was further supported by linkage analysis with 11q23 markers. Lod scores between A-O and these markers, cumulated over three large Arab families, were significant and confirmed the localization of the ATA gene to aq23. However, another Druze family unassigned to a specific complementation group, showed several recombinants between A-T and the same markers, leaving the localization of the A-T gene in this family open.     

Analysis of β-globin gene haplotypes in Asian Indians: origin and spread of β-thalassaemia on the Indian subcontinent

-globin gene haplotypes were determined for 196 normal (-A) and 419 thalassaemia (-Th) chromosomes of individuals from four different regions of the Indian subcontinent; North-west Pakistan, Gujarat, Punjab and Sindh. Analysis of -A and -Th haplotypes and haplotype-mutation associations in each regional group along with a consideration of Indian history provided information about the origin and spread of -thalassaemia mutations on the Indian subcontinent. The data are consistent with relatively recent and local origins for most -thalassaemia mutations. The frequencies of particular alleles differ markedly in various regions and these may be useful population markers. Of the high frequency alleles, intervening sequence 1 (IVS-1) nucleotide 5 (G-C) and codons 41/42 (-CTTT) appear to be older as suggested by multiple haplotype associations and a widespread geographical distribution. The microepidemiology of -thalassaemia in this region reflects considerable ethnic diversity, gene flow from population migration and natural selection by malaria infection.     

电损毁海马CA3区及连合前穹窿对大鼠血浆胰岛素水平...

Bilateral electrical lesioning of the hippocampal CA3 region (HCA3-EL) or anterior commissura hippocampi (ACHF-EL) caused marked elevations in plasma basal levels of insulin. 2 weeks later, fasting blood glucose levels were also augmented with decreased glucose tolerance. In contrast, the secretory response of pancreatic B cells to glucose stimulation was markedly enhanced. Following intravenous glucose tolerance test (IVGTT), the relative amounts of glucagon-like and insulin-like immunoreactants were reduced in the pancreatic islets of both HCA3-EL and ACHF-EL rats in comparison with the controls. In the HCA3-EL group, the relative amounts of somatostatin-like immunoreactants and gross numbers of such immunostained cells in islets were also decreased as compared with the control. No difference was seen in pancreatic-polypeptide-like immunoreactivities as assessed by immunohistochemistry plus microphotometry method. The above results suggest strongly that HCA3 and ACHF exert a tonic inhibitory action on the insulin secretion in the rat.     

从呼气末CO2浓度与动脉血压的关系判断人工呼吸猫...

End-tidal CO2 (ET-CO2) provides a continuous and sensitive monitoring of proper pulmonary ventilation in artificially ventilated animals during single unit recordings of the central nervous system. To determine a reliable standard of ET-CO2 in anesthetized and paralyzed animal, the relationship between ET-CO2 and arterial blood pressure (ABP) was observed in cat under different ventilation level. The results showed that ABP changed tremendously with variation in ventilation, but remained constant at a normal level as long as ET-CO2 was maintained within the range between 4.0-5.0%, i.e. optimally at 4.5 +/- 0.5%.     

Comparison of different procedures for the extraction of DNA from small blood samples of non-human primates

In this study three different techniques suitable to recover high molecular weight genomic DNA from small blood samples of different species of malagasy primates are compared: we suggest the use of a very simple phenol-chlorophorm DNA extraction for badly stored or coagulated blood as for samples collected under difficult field conditions. Furthermore we briefly describe the use of this DNA in determining RLFP patterns and DNA fingerprints.