Temporal and spatial specificity of PDGF alpha receptor promoter in transgenic mice

Aberrant expression of the platelet-derived growth factor alpha receptor (PDGF alpha R) has been linked to developmental abnormalities in vertebrate models, and has been implicated in multiple disease states in humans. To identify cis-acting regulatory elements that dictate expression of this receptor, we generated transgenic mice bearing the reporter gene beta-galactosidase (lacZ) under the control of a 6-kb promoter sequence. Expression of lacZ was monitored throughout embryonic development, with special focus on nervous tissue, skeleton, and several organ systems wherein PDGF alpha R expression is thought to play a pivotal role. In several independent transgenic mouse strains, lacZ expression recapitulated predominant features of PDGF alpha R gene expression during mouse development. These results demonstrate that critical tissue-specific regulatory elements for PDGF alpha R expression are located within a 6-kb upstream region of the PDGF alpha R gene.     

The relationship between the severity of sleep apnea syndrome and 24-h blood pressure values in patients with obstructive sleep apnea

Analysis of the nucleotide sequence in the 5' flanking region of bacteriophage T4 gene 25 revealed three potential Shine and Dalgarno sequences, SD1, SD2 and SD3, with a spacing of 8, 17 and 27 nucleotides from the initiation codon of this gene, respectively. Results of our experiments in the bacteriophage T7 expression system clearly demonstrate that the SD3 sequence is required for efficient expression of gene 25. We propose the existence of a stem-loop structure that includes SD1 and SD2 sequences and brings the SD3 sequence to a favourable spacing with the initiation codon of gene 25. Since the predicted secondary structure in the translational initiation region of gene 25 is relatively unstable and the SD3 sequence, GAGG, is more typical than the SD1 sequence, GAG, we suggest that this structure could control the level of gene expression.     

Genetic basis of human complement C8 alpha-gamma deficiency

Deficiency of the alpha-gamma subunit of the eighth component of complement (C8alpha-gammaD) is frequently associated with recurrent neisserial infections, especially meningitis caused by Neisseria meningitidis. We here report the molecular basis of C8alpha-gammaD in two unrelated Japanese subjects. Screening all 11 exons of the C8alpha gene and all 7 exons of the C8gamma gene and their boundaries by exon-specific PCR/single-strand conformation polymorphism demonstrated aberrant single-stranded DNA fragments in exon 2 of C8alpha gene in case 1 and in exons 2 and 9 of C8alpha gene in case 2. Nucleotide sequencing of the amplified DNA fragments in case 1 revealed a homozygous single-point mutation at the second exon-intron boundary, inactivating the universally conserved 5' splice site consensus sequence of the second intron (IVS2+1G-->T). Case 2 was a compound heterozygote for the splice junction mutation, IVS2+1G-->T, and a nonsense mutation at Arg394 (R394X). R394X was caused by a C to T transition at nucleotide 1407, the first nucleotide of the codon CGA for Arg394, leading to a stop codon TGA. No mutations were detected in the C8gamma gene by our method. Our results indicate that the pathogenesis of C8alpha-gammaD might be caused by heterogeneous molecular defects in the C8alpha gene.     

Isolation and molecular characterization of high-performance cellobiose-fermenting spontaneous mutants of ethanologenic Escherichia coli KO11 containing the Klebsiella oxytoca casAB operon

Escherichia coli KO11 was previously constructed to produce ethanol from acid hydrolysates of hemicellulose (pentoses and hexoses) by the chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB). Klebsiella oxytoca P2 was constructed in an analogous fashion for the simultaneous saccharification and fermentation of cellulose and contains PTS enzymes for cellobiose. In this study, KO11 was further engineered for the fermentation of cellulose by adding the K. oxytoca casAB genes encoding Enzyme IIcellobiose and phospho-beta-glucosidase. Although the two K. oxytoca genes were well expressed in cloning hosts such as DH5 alpha, both were expressed poorly in E. coli KO11, a derivative of E. coli B. Spontaneous mutants which exhibited more than 15-fold-higher specific activities for cellobiose metabolism were isolated. The mutations of these mutants resided in the plasmid rather than the host. Three mutants were characterized by sequence analysis. All contained similar internal deletions which eliminated the casAB promoter and operator regions and placed the lacZ Shine-Dalgarno region immediately upstream from the casA Shine-Dalgarno region. KO11 harboring mutant plasmids (pLOI1908, pLOI1909, or pLOI1910) rapidly fermented cellobiose to ethanol, and the yield was more than 90% of the theoretical yield. Two of these strains were used with commercial cellulase to ferment mixed-waste office paper to ethanol.     

Molecular cloning of human G alpha q cDNA and chromosomal localization of the G alpha q gene (GNAQ) and a processed pseudogene

G alpha q is the alpha subunit of one of the heterotrimeric GTP-binding proteins that mediates stimulation of phospholipase C beta. We report the isolation and characterization of cDNA clones from a frontal cortex cDNA library encoding human G alpha q. The encoded protein is 359 amino acids long and is identical in all but one amino acid residue to mouse G alpha q. Analysis of human genomic DNA reveals an intronless sequence with strong homology to human G alpha q cDNA. In comparison to G alpha q cDNA, this genomic DNA sequence includes several small deletions and insertions that alter the reading frame, multiple single base changes, and a premature termination codon in the open reading frame, hallmarks of a processed pseudogene. Probes derived from human G alpha q cDNA sequence map to both chromosomes 2 and 9 in high-stringency genomic blot analyses of DNA from a panel of human-rodent hybrid cell lines. PCR primers that selectively amplify the pseudogene sequence generate a product only when DNA containing human chromosome 2 is used as the template, indicating that the authentic G alpha q gene (GNAQ) is located on chromosome 9. Regional localization by FISH analysis places GNAQ at 9q21 and the pseudogene at 2q14.3-q21.     

A short burst of oxygen radicals at reflow induces sustained release of oxidized glutathione from postischemic hearts

Three different catalytic isoforms of cAMP-dependent protein kinase have been identified (C alpha, C beta, and C gamma). We report the cloning and characterization of the human and rhesus monkey genes encoding the testis-specific C gamma subunit. The human C gamma gene is intronless with an open reading frame similar to the previously published cDNA sequence. The 3' and 5' flanking regions share high similarity with the C alpha nontranslated regions (82%) also outside the regions corresponding to the C gamma cDNA. The human gene is flanked by an Alu-related sequence in the 5'-end and there are insertions of two Alu-related sequences in the 3' nontranslated region. The observation that the C gamma gene is intronless and colinear with C alpha mRNA, together with the presence of remnants of a poly(A) tail and flanking direct repeats, indicates that the C gamma gene is a C alpha-derived retroposon. The 5' flanking region of this gene has a high G/C content and a putative TATA box situated at -138 compared to the translation initiation codon. Cloning and sequencing of a partial C gamma rhesus monkey gene demonstrate conservation of the sequence in primates. Northern analysis on isolated and fractionated human germ cells of testes from normal and estrogen-treated individuals demonstrates that the C gamma gene is expressed only in germ cells in the human testis. Our results indicate that the C gamma gene is a retroposon specifically transcribed in primate testicular germ cells.     

Characterization of the pUC19-lacZC141 reversion system for assaying chemical mutagenesis

pUC19-lacZC141 DNA contains a proline codon at positions 141 to 143, where an alanine codon normally appears in the original lacZ gene. pUC19-lacZC141 DNA was produced using site-directed mutagenesis. After transfection of pUC19-lacZC141 DNA into lacZ hosts, the transformants produce white colonies on an agar plate containing X-gal and IPTG. lacZ+ revertants can be identified by their dark- and light-blue colony color against a background of non-mutant white colonies, indicating restoration of beta-galactosidase activity. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methylmethanesulfonate (MMS) were used to characterize the pUC19-lacZC141 DNA reversion assay. Mutagenesis resulting from methylated DNA was examined in Escherichia coli strains JM109, BMH71-18mutS, and SURE, which differ in their repair systems for DNA damage. In JM109 and BMH71-18mutS, mostly G:C-->A:T transitions and some G:C-->C:G or G:C-->T:A transversions were observed. E. coli SURE produced, in addition, frameshift mutations (approximately 10%). The DNA sequence analysis of 174 induced mutants indicated that the major effect of methylation is on single base-pair substitutions with a slight effect on deletion frameshifts. All mutations are consistent with miscoding of guanine or cytosine adducts or lesions. Transitions account for 158 of 165 (96%) induced base substitutions. Approximately 93% of the base-substitution mutations occurred at the expected positions 141 to 143 in the lacZ gene. The pUC19-lacZC141 assay was sufficiently sensitive to allow the detection of mutations in lacZ- hosts with different repair mechanisms. The pUC19-lacZC141 DNA reversion system will permit the assaying of other chemicals not otherwise amenable to mutagenesis studies.     

Molecular cloning and nucleotide sequence of an endo-1,5-alpha-L-arabinase gene from Bacillus subtilis

The nucleotide sequence of the gene encoding an endo-1,5-alpha-L-arabinase (protopectinase C) of Bacillus subtilis was determined by sequencing fragments amplified by the cassette-ligation-mediated PCR (CLM-PCR). The gene covering the start and stop codon was amplified by PCR with two specific primers, which were designed from the sequence data determined by CLM-PCR. An approximately 1.5-kb amplification product was cloned into the vector pUC119, forming a plasmid termed pPPC. An ORF that encodes the arabinase composed of 324 amino acids including a 33-amino-acid signal peptide was assigned. Comparison of the deduced amino acid sequence of the enzyme with that of an Aspergillus niger endoarabinase showed 37% identity in a 207-amino-acid overlap. The optimal nucleotide sequence for catabolite repression of B. subtilis was found upstream of the structural gene. In a culture of Escherichia coli DH5alpha cells harboring pPPC, no arabinase activity was detected, either intracellularly or extracellularly, suggesting that the B. subtilis promotor is not functional in this transformant. In B. subtilis IFO 3134 strain, production of protopectinase C was repressed by readily metabolizable carbohydrates. In contrast, productivity (total enzyme activity/bacterial growth) of the enzyme was increased about fourfold in the presence of 0.75 M potassium phosphate in the culture medium. The phosphate anion seemed to be involved in the stimulation of protopectinase C production in this stain.