NCR-1 and NCR-2, the C. elegans homologs of the human Niemann-Pick type C1 disease protein, function upstream of DAF-9 in the dauer formation pathways

Mutations in the human NPC1 gene cause most cases of Niemann-Pick type C (NP-C) disease, a fatal autosomal recessive neurodegenerative disorder. NPC1 is implicated in intracellular trafficking of cholesterol and glycolipids, but its exact function remains unclear. The C. elegans genome contains two homologs of NPC1, ncr-1 and ncr-2, and an ncr-2; ncr-1 double deletion mutant forms dauer larvae constitutively (Daf-c). We have analyzed the phenotypes of ncr single and double mutants in detail, and determined the ncr gene expression patterns. We find that the ncr genes function in a hormonal branch of the dauer formation pathway upstream of daf-9 and daf-12, which encode a cytochrome P450 enzyme and a nuclear hormone receptor, respectively. ncr-1 is expressed broadly in tissues with high levels of cholesterol, whereas expression of ncr-2 is restricted to a few cells. Both Ncr genes are expressed in the XXX cells, which are implicated in regulating dauer formation via the daf-9 pathway. Only the ncr-1 mutant is hypersensitive to cholesterol deprivation and to progesterone, an inhibitor of intracellular cholesterol trafficking. Our results support the hypothesis that ncr-1 and ncr-2 are involved in intracellular cholesterol processing in C. elegans, and that a sterol-signaling defect is responsible for the Daf-c phenotype of the ncr-2; ncr-1 mutant.     

Direct immunological identification of full-length cDNA clones for plant protein without gene fusion to E. coli protein

By immunological screening of a cDNA library constructed from potato tuber poly(A)+ RNA and Escherichia coli expression vector pUC8 by the vector-primer and linker procedure of Okayama and Berg [(1982) Mol. Cell Biol. 2, 161-170], nearly full-length cDNA clones for patatin, a major protein of potato tuber, were identified. The cDNA carrying part of the 5'-noncoding region of the patatin mRNA, in addition to entire coding and 3'-noncoding regions, expressed prepatatin in E. coli cells by translational initiation inside cDNA. These results suggest that nearly full-length cDNA clones with entire coding region can be identified directly by immunological screening without gene fusion to E. coli proteins at least for some plant mRNAs.     

Structural feature of sorghum chloroplast psbA gene and regulation effects of its 5'-noncoding region*

Comparative analysis reveals remarkable homology between the sequences of both psbA gene nucleotides and the inferred amino acids of sorghum, a C_4 plant, and those of rice, a C_3 plant. The 5'-noncoding region of sorghum psbA gene contains the conservative promoter elements, "-35" element and "-10" element, like the prokaryote and the promoter element, TATA box, like the eukaryote. As compared with that of the rice, an extra sequence of 7 bp is found in the leader sequence of the mRNA in the former. Using an in vitro system, it has been demonstrated that protein factor exists in sorghum chloroplast protein extract which specifically binds to the 5'-noncoding region of psbA gene. Measurement of the expression of luciferase shows a 2—5 time greater reaction of the expression plasmids pALqs which contain leader region of sorghum psbA gene than that of the expression plasmids pALqr which contain leader region of rice psbA gene in E. coli.     

Cloning and nucleotide sequence of the simian rotavirus gene 6 that codes for the major inner capsid protein.

The nucleotide sequence of the gene that codes for the major inner capsid protein of the simian rotavirus SA11 has been determined. A DNA copy of mRNA from gene 6 was cloned in the E. coli plasmid pBR322. The full-length gene is 1357 nucleotides long with a 5'-noncoding region of 23 nucleotides and a 3'-noncoding region of 140 nucleotides. The gene contains a single, long, open reading-frame of 1194 nucleotides capable of coding for a protein of 397 amino acids with a molecular weight of 44,816. The predicted protein product is relatively proline-rich with a net charge at neutral pH of -3.5. One stretch of 53 amino acids (encoded by nucleotides 327-485) is basic.     

人β神经生长因子基因稀有密码子及mRNA二级结构对其在大肠杆菌中表达的影响

目的:研究人β神经生长因子(β-NGF)基因中稀有密码子及其mRNA二级结构对其在大肠杆菌中表达量的影响。方法:根据对人β-ngf中稀有密码子及其mRNA二级结构的研究,同义突变人β-ngf基因,通过PCR得到人β-ngf的5’端同义突变基因rh-β-ngfp32和全同义突变基因rh-β-ngfmu,将这2个序列克隆入载体pET3a中,得到重组质粒pET3a-ngfp32和pET3a-ngfmu,分别转化大肠杆菌BL21(DE3)感受态,IPTG诱导表达,收集菌体,SDS-PAGE检测其表达量的改变。结果:构建的pET3a-ngfp32和pET3a-ngfmu表达载体酶切和测序结果正确,SDS-PAGE结果显示,与在重组菌pET3a-NGF总蛋白中的表达量相比,目的蛋白rh-β-NGF在重组菌pET3a-NGFP32和pET3a-NGFmu中的表达量均明显增高,并且在重组菌pET3a-NGFmu中的表达量高于重组菌pET3a-NGFP32。结论:目的蛋白rh-β-NGF在重组菌pET3a-NGFP32和pET3a-NGFmu中表达量的增高,说明人β-ngf基因中稀有密码子和mRNA的二级结构对其在大肠杆菌中的表达有较为明显的影响,结果为构建rh-β-NGF的大肠杆菌工程菌株奠定了基础。     

Expression of glutathione peroxidase I gene in selenium-deficient rats.

We have characterized a cDNA pGPX1211 encoding rat glutathione peroxidase I. The selenocysteine in the protein corresponded to a TGA codon in the coding region of the cDNA, similar to earlier findings in mouse and human genes, and a gene encoding the formate dehydrogenase from E. coli, another selenoenzyme. The rat GSH peroxidase I has a calculated subunit molecular weight of 22,155 daltons and shares 95% and 86% sequence homology with the mouse and human subunits, respectively. The 3'-noncoding sequence (greater than 930 bp) in pGPX1211 is much longer than that of the human sequences. We found that glutathione peroxidase I mRNA, but not the polypeptide, was expressed under nutritional stress of selenium deficiency where no glutathione peroxidase I activity can be detected. The failure of detecting any apoprotein for the glutathione peroxidase I under selenium deficiency and results published from other laboratories supports the proposal that selenium may be incorporated into the glutathione peroxidase I co-translationally.     

Structure and restriction fragment length polymorphism of genes for human liver arylamine N-acetyltransferases.

Genomic DNA clones coding for polymorphic and monomorphic arylamine N-acetyltransferases (NAT) of human liver were isolated from a genomic DNA library, and their restriction maps and partial nucleotide sequences were determined. Messenger RNA for monomorphic NAT was coded in one exon, while mRNA for polymorphic NAT was coded in two exons; the 5'-noncoding region was located in one exon 8 kb upstream from another exon containing the coding and 3'-noncoding regions. Recently, we have shown that there are three types of polymorphic NAT gene; one of the genes corresponds to a high NAT activity, while the other two genes give rise to a low NAT activity. The restriction fragment length polymorphism (RFLP) was analyzed by Southern blot hybridization of genomic DNAs from homozygotes of the three polymorphic NAT genes using various fragments of the cloned NAT gene. RFLPs of polymorphic NAT gene were observed in coding and 3'-flanking region upon digestion with BamHI and KpnI.     

Nucleotide sequence of the chicken cardiac alpha actin gene: absence of strong homologies in the promoter and 3'-untranslated regions with the skeletal alpha actin sequence

The entire nucleotide sequence of the chicken cardiac alpha-actin (CC alpha A) gene has been determined. This is the first complete sequence of a cardiac actin gene that includes the promoter region, cap site, all the introns, and the polyadenylation site. The gene contains six introns, five of which interrupt the coding region at amino acids (aa) 41, 150, 204, 267, and 327. The first intron is in the 5'-noncoding region and is 438 bp in length. The CC alpha A gene encodes an mRNA of approx. 1400 bp with 5'- and 3'-untranslated region of 59 and 184 nucleotides (nt), respectively. Like the chicken skeletal alpha-actin gene, the CC alpha A gene has the codon for the aa cysteine between the initiator ATG and the codon for the N-terminal aspartic acid residue of the mature protein. There are no strong homologies (less than 13 consecutive nt) in the promoter or 3'-untranslated regions between the CC alpha A and chicken skeletal alpha-actin genes even though both are expressed in skeletal muscle during development. However, the 3'-untranslated region of the CC alpha A gene demonstrates significant sequence homology (76% over a 200-nt region) with the same region in the partial sequence of the human cardiac gene. The conservation of these sequence homologies between identical isoforms rather than the different alpha actin genes suggests these conserved regions may have a role in regulation rather than tissue-specific expression, as previously proposed.     

Construction of a plasmid for high-level expression of goat alpha-lactalbumin and its derivative in E. coli

The high-level expression system of goat alpha-lactalbumin (alpha-LA) in E. coli was established by fusing the alpha-LA cDNA to porcine adenylate kinase cDNA and expressing the fused gene under the control of tac promoter. For high-level expression, elimination of 3'-noncoding region of the alpha-LA cDNA was found to be necessary.     

An improved Escherichia coli expression vector for the construction and identification of full-length cDNA clones

An Escherichia coli expression vector designed for the efficient synthesis and identification of a full-length cDNA clone is constructed. The vector allows the synthesis of double-stranded cDNAs downstream from the tandem lac control regions employing the vector-primer and linker procedure of Okayama and Berg [Mol. Cell Biol. 2 (1982) 161-170]. Full-length cDNA clones carrying the 5'-noncoding region in addition to the entire coding and 3'-noncoding regions can be expressed in E. coli cells without fusing their coding region to that of E. coli proteins; these clones are identified by colony immunoassay. The entire cDNA insert can be easily excised from the plasmid, since the multiple cloning sites in the vector are duplicated at both ends of the cDNA insert during its synthesis.     

Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa.

We have isolated and characterized a gene, fdh, from Neurospora crassa which is developmentally regulated and which produces formate dehydrogenase activity when expressed in Escherichia coli. The gene is closely linked (less than 0.6 kb apart) to the leu-5 gene encoding mitochondrial leucyl-tRNA synthetase; the two genes are transcribed convergently from opposite strands. The expression patterns of these genes differ: fdh mRNA is found only during conidiation and early germination and is not detectable during mycelial growth, while leu-5 mRNA appears during germination and mycelial growth. The structure of the fdh gene was determined from the sequence of cDNA and genomic DNA clones and from mRNA mapping studies. The gene encodes a 375-amino-acid-long protein with sequence similarity to NAD-dependent dehydrogenases of the E. coli 3-phosphoglycerate dehydrogenase (serA gene product) subfamily. In particular, there is striking sequence similarity (52% identity) to formate dehydrogenase from Pseudomonas sp. strain 101. All of the residues thought to interact with NAD in the crystal structure of the Pseudomonas enzyme are conserved in the N. crassa enzyme. We have further shown that expression of the N. crassa gene in E. coli leads to the production of formate dehydrogenase activity, indicating that the N. crassa gene specifies a functional polypeptide.     

Comparison of the lipoprotein gene among the enterobacteriaceae. DNA sequence of Morganella morganii lipoprotein gene and its expression in Escherichia coli

A DNA sequence of 532 base pairs encompassing the entire Morganella morganii lipoprotein gene (lpp) was determined. Sequence comparisons of the M. morganii lpp gene with the lpp genes from Escherichia coli, Serratia marcescens, and Erwinia amylovora reveal that the M. morganii lpp gene is more distantly related to the E. coli lpp gene than any of the other lpp genes examined. Between the E. coli and M. morganii lpp genes, the following homologies were found: 44% in the promoter region (bases, -45 to -1), 88% in the 5'-end untranslated region of the mRNA, 58% in the signal sequence coding region, 75% in the coding region for the first 51 and 43% for the last 7 amino acid residues. Upstream of the promoter region and downstream of the termination codon, there are extensive insertions, deletions, and base substitutions. In spite of the differences in the DNA sequences, the lipoprotein structure was found to be highly conserved except for the carboxyl-terminal sequence of 7 amino residues. The coding region of the M. morganii lpp gene including the signal sequence was inserted into an expression cloning vector so that the production of the M. morganii lipoprotein could be induced in E. coli by a lac inducer, isopropyl-beta-D-thioglactoside. It was found that when induced, the M. morganii prolipoprotein was apparently secreted normally across the E. coli cytoplasmic membrane, modified with glycerol and palmitic acid, processed to the mature lipoprotein, and assembled in the E. coli outer membrane. The bound form covalently linked to the peptidoglycan was also found.     

Gene structure of Enterococcus hirae (Streptococcus faecalis) F1F0-ATPase, which functions as a regulator of cytoplasmic pH.

Enterococcus hirae (formerly Streptococcus faecalis) ATCC 9790 has an F1F0-ATPase which functions as a regulator of the cytoplasmic pH but does not synthesize ATP. We isolated four clones which contained genes for c, b, delta, and alpha subunits of this enzyme but not for other subunit genes. It was revealed that two specific regions (upstream of the c-subunit gene and downstream of the gamma-subunit gene) were lost at a specific site in the clones we isolated, suggesting that these regions were unstable in Escherichia coli. The deleted regions were amplified by polymerase chain reaction, and the nucleotide sequences of these regions were determined. The results showed that eight genes for a, c, b, delta, alpha, gamma, beta, and epsilon subunits were present in this order. Northern (RNA) blot analysis showed that these eight genes were transcribed to one mRNA. The i gene was not found in the upper region of the a-subunit gene. Instead of the i gene, this operon contained a long untranslated region (240 bp) whose G + C content was only 30%. There was no typical promoter sequence such as was proposed for E. coli, suggesting that the promoter structure of this species is different from that of E. coli. Deduced amino acid sequences suggested that E. hirae H(+)-ATPase is a typical F1F0-type ATPase but that its gene structure is not identical to that of other bacterial F1F0-ATPases.