Molecular analysis of the murine C4b-binding protein gene. Chromosome assignment and partial gene organization

Murine C4b-binding protein (C4BP) is a regulatory molecule in the classical pathway of complement. C4BP is composed predominantly of short consensus repeats (SCRs) approximately 60 amino acids in length, which contain a framework of conserved residues. The SCRs are found in many complement molecules and a growing number of noncomplement molecules as well and are a major structural feature of some of these molecules. To characterize the structure of the murine C4BP gene, a cosmid library constructed from Balb/c liver DNA was screened. Several nearly identical, overlapping clones were identified; however, none of the clones, alone or in combination, covered the entire C4BP gene. One clone (D26) was chosen for detailed analysis and found to contain all but the leader region, the first SCR, and the first half of the second SCR. The SCRs three through six were each encoded by single exons. Only the latter half of the second SCR was present on the clone, and it was encoded by a single exon, demonstrating that murine C4BP has a split SCR at the genomic level. Structural mapping of this portion of the gene demonstrates that the region extending from the second half of the second SCR through the nonrepeat and untranslated region spans approximately 12 kb; however, genomic Southern blot analysis suggests that the gene is between 20 and 30 kb in length. Analysis of the 3' genomic sequence demonstrates that this region of the gene has homology with SV-40 late (class II) RNA sequences. These sequences may play a role in 3' cleavage of the precursor RNA prior to polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genomic organization of the gene and a related pseudogene for a human folate binding protein.

An unprocessed pseudogene which is 90% homologous with the cDNA encoding a folate binding protein in KB cells has been cloned from a human genomic library. This pseudogene contains TGA stop codons, base deletions and substitutions and lacks a 5' region. The size of the exons and the intron-exon sites are almost identical to the organization of the gene encoding this protein which has now been characterized from genomic DNA using the polymerase chain reaction with selected primers to the cDNA.     

Structural analysis of the locus containing the human C-reactive protein gene and its related pseudogene

The gene for human C-reactive protein (CRP) is mapped within a 34-kilobase pair genomic DNA segment identified by chromosome walking through overlapping DNA fragments cloned into a lambda phage library. Within 16 kilobase pairs upstream and downstream of the locus for the authentic CRP gene, only one other sequence homologous to that for CRP could be found. Sequencing analysis indicates this sequence to be a pseudogene with 50-80% region-specific homology. Comparison of the authentic CRP gene cloned from genomic DNA libraries independently prepared from three patients indicates no difference in the 5' and 3' flanking region, promoter region, or coding sequence. Only a polymorphism in the length of the poly(GT) stretch located in the intron is observed. There appears to be only one gene locus and copy per haploid chromosome for the authentic CRP gene and its pseudogene.     

一个与小鼠锌指蛋白基因ZF—12相关的假基因的克隆和鉴定

小鼠类Krueppel锌指蛋白基因ZF-12为人的类Krueppel锌指蛋白基因ZNF191的同源基因,它们都编码368个氨基酸残基,N端存在SCAN结构域,C端有4个连续的锌指模体,最近的研究表明ZNF191是肝癌发生的相关基因,我们以人锌指蛋白基因ZNF191的vDNA为探针,筛选小鼠λ噬菌体基因组文库,意外地获得了1个与小鼠锌指蛋白基因ZF-12相类似的基因,多种组织的RT-PCR和启动子序列分析,暗示该基因不表达,且该基因无内含子,与ZF-12高度相似,存在突变,暗示其为与ZF-12相关的假基因序列,经查新证实它为新的序列后,以ZF12p（ZF-12pseudogene)命名在GenBank登录（AY040222）。查录GenBank的人类基因组库以及Southern结果显示人类基因组中无ZNF191假基因序列,ZF12p与ZF-12高度相似,暗示ZF12p在进化过程中产生的时间较晚,这对研究锌指蛋白基因ZF-12的突变与进化具有重要的意义。     

Cloning and genomic organization of the mouse gene slc23a1 encoding a vitamin C transporter.

Vitamin C is known to exist in particularly high concentrations in brain tissue, and its free radical scavenging function is thought to represent a major antioxidative defense system. We have cloned, sequenced and analyzed the genomic structure of a mouse sodium-dependent vitamin C transporter gene, Slc23a1 (also known as Svct2). The mouse Slc23a1 cDNA is 6.4 kb long and was cloned directly from a mouse brain RNA preparation. Hybridization screening of a mouse genomic BAC library identified BAC 53L21 which contains at least the entire coding sequence of the mouse Slc23a1 gene. Determination of the exon-intron structure of the gene revealed 17 exons ranging from 58 bp to 4407 bp extending over 50 kb of the mouse genome, with the translation start codon located in exon 3. Its 1944 nucleotide open reading frame encodes a polypeptide of 647 aa, which is highly similar to rat and human orthologs. The mouse gene was assigned to chromosome 2qG2 by fluorescence in situ hybridization analysis. Expression of this gene was demonstrated in a wide range of tissues, with especially high levels in brain. Neurodegenerative diseases with an established role for oxidative stress in the cytoplasm may therefore be conditions of SLC23A1 dysfunction. Key words: gene structure; Vitamin C; transporter; oxidative stress     

Cloning and sequencing of the gene for the DNA-binding 17 K protein of Escherichia coli

The skp gene encoding the 17 K protein, a basic DNA-binding nucleoid-associated protein of Escherichia coli, was cloned as part of a 2.3-kb genomic fragment. The gene was sequenced and a polypeptide of 161 amino acids (aa) was deduced from the nucleotide sequence. The primary translation product was processed by cutting off the N-terminal 20 aa residues, yielding a mature polypeptide of 141 aa. The M_r of the mature polypeptide was 15674. An E. coli transformant containing the skp gene on the plasmid pGAH317 was shown to overproduce the gene product some 20-fold.     

Evidence for vitamin K semiquinone as the functional form of vitamin K in the liver vitamin K-dependent protein carboxylation reaction.

The protein carboxylating system derived from vitamin K-deficient rat liver microsomes functions in detergent solution if vitamin K₁, NADH, dithiothreitol, CO₂ and O₂ are added. The requirements for added NADH, dithiothreitol and O₂ are all eliminated by the use of vitamin K₁ hydroquinone in place of quinone. The use of the hydroquinone gives a more rapid reaction and a higher yield than does the quinone plus reducing system. The reaction proceeding from either the vitamin K₁ quinone or hydroquinone is blocked by the spin-trapping agent, 5,5-dimethyl-l-pyrroline-N-oxide, suggesting that the active form of vitamin K is the semiquinone.     

N-bromoacetyl-peptide substrate affinity labeling of vitamin K dependent carboxylase.

Vitamin K dependent carboxylase (carboxylase) is a membrane-associated endoplasmic reticular enzyme that catalyzes the conversion of certain glutamate residues of essential blood coagulation proteins to gamma-carboxyglutamyl (Gla) residues. A series of N-bromoacetyl-peptide substrate affinity labels based on the Gla domain of these blood-clotting proteins was synthesized, and the substrate and inactivator kinetic parameters were assessed. The most promising of these affinity peptides, N-bromoacetyl-FLEELY, was both substrate for carboxylase and an irreversible time-dependent inactivator of the enzyme, inactivating 80% of carboxylase under pseudo-first-order conditions. Addition of saturating amounts of a competing peptide substrate completely abolished the inhibitory properties of N-bromoacetyl-FLEELY, consistent with inactivation occurring at the active site. The partition ratio of inactivation/carboxylation was 1/30. The 94-kDa carboxylase was purified to 15-50% purity by a modification of a recent protocol [Wu, S.-M., Morris, D. P., & Stafford, D. W. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2236-2240] and covalently labeled with N-bromoacetyl-FLEEL[125I]Y. On silver-stained 10% sodium dodecyl sulfate-polyacrylamide gels, the predominant radiolabeled band was the 94,000 molecular weight species. This result independently validates that the 94-kDa protein is a carboxylase.     

Cloning and expression of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli.

The transport of cyanocobalamin (vitamin B12) in cells of Escherichia coli is dependent on a receptor protein (BtuB protein) located in the outer membrane. A 9.1-kilobase pair BamHI fragment carrying the btuB gene was cloned from a specialized transducing phage into multicopy plasmids. Insertions of transposon Tn1000 which prevented production of the receptor localized btuB to a 2-kilobase pair region. Further subcloning allowed isolation of this region as a 2.3-kilobase pair Sau3A fragment. The BtuB+ plasmids were shown by maxicell analysis to encode a polypeptide with a molecular weight of 66,000 in the outer membrane. This polypeptide was missing in cells with Tn1000 insertions in btuB and was reduced in amount upon growth of plasmid-bearing cells in repressing concentrations of vitamin B12. Several Tn1000 insertions outside the 5' end of the coding region exhibited reduced production of receptor. A deletion at the 3' end of btuB resulted in formation of an altered receptor. Amplified production of this polypeptide was associated with increased levels of binding of the receptor's ligands (vitamin B12 and phage BF23), increased rates of vitamin B12 uptake, and altered susceptibility to the group E colicins. Deficiency in various major outer membrane proteins did not affect production of the btuB product, and the amplified levels of this protein partially reversed the tolerance to E colicins seen in these mutants.     

Molecular cloning and sequence analysis of the mouse kallikrein-binding protein gene.

A genomic clone (MKBP-10) encoding the mouse kallikrein-binding protein (MKBP) was isolated from a mouse genomic DNA library by screening with a rat kallikrein-binding protein (RKBP) cDNA probe. The total sequenced region of the MKBP gene spans 8615 base pairs. The exon and intron locations of the RKBP gene were identified by similarity with the RKBP gene. The MKBP gene encodes a prepeptide of 417 amino acid residues which exhibits 71% homology with RKBP. A TATA box sequence was located in the 5' flanking region of the MKBP gene by similarity with the consensus sequence TATAAAA.