Molecular Epidemiology and Diagnosis of PBG Deaminase Gene Defects in Acute Intermittent Porphyria

Acute intermittent porphyria (AIP) is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding for porphobilinogen (PBG) deaminase and is characterized by life-threatening neurovisceral attacks, often precipitated by drugs, fasting, cyclical hormonal changes, or infectious diseases. This report describes a prospective study on the molecular epidemiology of PBG deaminase gene defects in AIP. It uses a sensitive, reliable, and easy-to-handle method for routine AIP molecular diagnosis and family study based on an exon-by-exon denaturing gradient gel electrophoresis (DGGE) strategy followed by direct sequencing. Fifteen genomic DNA fragments, including all the coding sequence and covering 3.35 kb of the PBG deaminase gene, were investigated in 405 subjects from 121 unrelated French Caucasian AIP families who had not been screened previously at the DNA level. PBG deaminase gene mutations were identified in 109 families, but only 78 were of different type, and each of them had a prevalence rate < 5%. Among these mutations, 33 had not been published previously. Sixty percent of these 78 mutations were located in only three exons (exons 10, 12, and 14), 44% were missense, 18% were splice defect, 19% were frameshift, and 16% were nonsense. In addition, two de novo mutational events were characterized. The evaluation of the efficiency of the standard PBG deaminase enzymatic screening method for gene-carrier detection indicated 95% of concordancy with the molecular-based diagnosis.     

Mutants of Escherichia coli K12 accumulating porphobilinogen: a new locus, hemC.

Mutants of Escherichia coli K12 which accumulated the haem precursor porphobilinogen are described. The mutants grew very slowly on carbon and energy sources which K12 uses only oxidatively, and they had low catalase activities, suggesting that they were deficient in haem. Extracts had one-tenth of the parental activity of the enzyme porphobilinogen deaminase. In transduction, the mutation mapped close to genes ilvD and metE at minute 84. The gene was tentatively identified as hemC, coding for porphobilinogen deaminase. The gene symbol hemC replaces the earlier and temporary symbol popE.     

Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.     

Structure and expression of chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.) isolated by redundant polymerase chain reaction.

Porphobilinogen (PBG) deaminase catalyzes the polymerization of four PBG monopyrrole units into the linear tetrapyrrole hydroxymethylbilane necessary for the formation of chlorophyll and heme in plant cells. Degenerate oligonucleotide primers were designed based on amino acid sequence data (generated by mass spectrometry) for purified PBG deaminase from pea (Pisum sativum L.) chloroplasts. These primers were used in TaqI polymerase-catalyzed polymerase chain reaction (PCR) amplification to produce partial cDNA and nuclear genomic fragments encoding the enzyme. Subsequently, a 1.6-kb cDNA was isolated by screening a cDNA library constructed in lambda gt11 from leaf poly(A)+ RNA with the PCR products. The cDNA encodes an approximately 40-kD polypeptide containing a 46-amino acid NH2-terminal transit peptide and a mature protein of 323 amino acids. The deduced amino acid sequence of the mature pea enzyme is similar to PBG deaminases from other species and contains the conserved arginine and cysteine residues previously implicated in catalysis. Northern blot analysis indicates that the pea gene encoding PBG deaminase is expressed to varying levels in chlorophyll-containing tissues and is subject to light induction.     

On the process of cellular division in Escherichia coli: nucleotide sequence of the gene for penicillin-binding protein 3

We determined the nucleotide sequence of a DNA fragment containing the ftsI gene coding for the penicillin-binding protein 3 (PBP-3), an indispensable enzyme for cell division of Escherichia coli. The entire ftsI gene was within the 2.8 kilobase PvuII fragment derived from the chromosomal segment on pLC26-6 (Nishimura et al. 1977). The coding region for PBP-3 was identified by comparison with the N-terminal amino acid sequence of in vitro synthesized PBP-3. The structural gene for ftsI consisted of 1,764 base-pairs coding for a 588 amino acid residue-polypeptide with a molecular weight of 63,850. PBP-3 synthesized in vitro showed a lower mobility in SDS-gel electrophoresis than that of the authentic PBP-3, suggesting that the primary translation product of the ftsI gene may be processed to yield mature PBP-3.     

Nucleotide sequence of a type II DNA topoisomerase gene. Bacteriophage T4 gene 39.

T4 DNA topoisomerase is a type II enzyme and is thought to be required for normal T4 DNA replication T4 gene 39 codes for the largest of the three subunits of T4 DNA topoisomerase. I have determined the nucleotide sequence of a region of 2568 nucleotides of T4 DNA which includes gene 39. The location of the gene was established by the identification of the first fifteen amino acids in the large open reading frame in the DNA sequence as those found at the amino-terminus of the purified 39-protein. The coding region of gene 39 has 1560 bases, and it is followed by two in-frame stop codons. The gene is preceded by a typical Shine-Dalgarno sequence as well as possible promoter sequences for E. coli RNA polymerase. T4 39-protein consists of 520 amino acids, and it has a calculated molecular weight of 58,478. By comparing the amino acid sequences, T4 39-protein is found to share homology with the gyrB subunit of DNA gyrase. This suggests that these topoisomerase subunits may be equivalent functionally. Some of the characteristics of the 39-protein and its structural features predicted from the DNA sequence data are discussed.     

Assembly of the mitochondrial membrane system. Sequence of the oxi 2 gene of yeast mitochondrial DNA

The region of mitochondrial DNA (mtDNA) containing the oxi 2 locus has been sequenced in a rho- clone (DS40) derived from the respiratory competent strain D273-10B/A48 of Saccharomyces cerevisiae. The DS40 clone was established to have retained only genetic markers in the oxi 2 locus and to have a segment of mtDNA extending from 18.6 to 24.3 units of the wild type map. The mitochondrial genome of DS40 includes a sequence that has been tentatively identified as the structural gene of Subunit 3 of cytochrome oxidase. The coding sequence is 810 nucleotides long and generates a protein with a molecular weight of 30,340. The amino acid composition of the oxi 2 gene product deduced from the nucleotide sequence is in agreement with the composition of the purified Subunit 3 of yeast cytochrome oxidase. The orientation of the DS40 mtDNA segment relative to wild type mtDNA indicates that the oxi 2 gene is transcribed from the same DNA strand as the oxi 1 and several other mitochondrial genes.     

DNA sequences of the D-serine deaminase control region and N-terminal portion of the structural gene

We determined the DNA sequence of the D-serine deaminase promoter region and of the N-terminal region of the structural gene. There are possibilities in the promoter for secondary structure and for initiation recognition sequences, and there is an open reading frame. The N-terminal sequence for the structural gene confirms that part of the amino acid sequence previously determined by E. Schlitz and W. Schmitt (FEBS Lett. 134:57-62, 1981), including the active site of the enzyme, and spans the two regions unresolved by their work.     

Determination of the nucleotide sequence for the exonuclease I structural gene (sbcB) of Escherichia coli K12

The complete nucleotide sequence of the structural gene for Escherichia coli exonuclease I has been determined. The coding region corresponds to a 465-amino acid protein with molecular weight of 53,174. The partial amino acid sequence of purified exonuclease I agrees with that predicted by the DNA sequence. Two putative weak promoters have been localized by S1 nuclease analysis. The sbcB coding sequence contains many non-optimal codons, characteristic of many poorly expressed E. coli genes.     

The primary structure of phosphoenolpyruvate carboxylase of Escherichia coli. Nucleotide sequence of the ppc gene and deduced amino acid sequence

The nucleotide sequence of the ppc gene, the structural gene for phosphoenolpyruvate carboxylase [EC 4.1.1.31], of Escherichia coli K-12 was determined. The gene codes for a polypeptide comprising 883 amino acid residues with a calculated molecular weight of 99,061. The amino acid sequence deduced from the nucleotide sequence was entirely consistent with the protein chemical data obtained with the purified enzyme, including the NH2- and COOH-terminal sequences and amino acid composition. The coding region is preceded by two putative ribosome binding sites, and is followed closely by a good representative of rho-independent terminator. The codon usage in the ppc gene suggests a moderate expression of the gene. The secondary structure of the enzyme was predicted from the deduced amino acid sequence.     

Overexpression and purification of asparagine synthetase from Escherichia coli.

Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUC18, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15% of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.     

Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain

A gene encoding cobalamin-dependent methionine synthase (EC 2.1.1.13) has been isolated from a plasmid library of Escherichia coli K-12 DNA by complementation to methionine prototrophy in an E. coli strain lacking both cobalamin-dependent and -independent methionine synthase activities (RK4536:metE, metHH). Maxicell expression of a series of plasmids containing deletions in the metH structural gene was employed to map the position and orientation of the gene on the cloned DNA fragment. A 6.3-kilobase EcoRI-SalI fragment containing the gene was cloned into the sequencing vector pGEM3B for double-stranded DNA sequencing; the MetH coding region consists of 3372 nucleotides. The enzyme was purified from an overproducing strain of E. coli harboring the recombinant plasmid, in which the level of methionine synthase was elevated 30- to 40-fold over wild-type E. coli. Recombinant enzyme is a protein of 123,640 molecular weight and has a turnover number of 1,450 min-1 in the standard assay. These values are to be compared with previously reported values of 133,000 for the molecular weight and 1,240-1,560 min-1 for the turnover number of the homogenous enzyme purified from a wild-type strain of E. coli B (Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H., and Matthews, R. G. (1988) Biochemistry 27, 8458-8465). Limited proteolysis of the native enzyme with trypsin resulted in loss of enzyme activity but retention of bound cobalamin on a peptide fragment of 28,000 molecular weight. This fragment has been shown to extend from residue 643 to residue 900 of the 1124-residue deduced amino acid sequence.     

稻瘟净（Blasticidin S）脱氨酶基因的克隆

从一株抗稻瘟净（ＢＳ）的Ａｓｐｅｒｇｉｌｌｕｓ ｔｅｒｒｅｕｓ菌中克隆到一个ｂｌａｓｔｉｅｉｄｉｎＳ脱氨酶基因,命名为ｂｓｒＡＳ。ＤＮＡ序列分析表明ｂｓｒＡＳ不含内含子。编码区长３９０ｂｐ,编码１３０个氨基酸。将ｂｓｒＡＳ转化到稻瘟菌中,能使受体菌表达出ＢＳ脱氨酶的活性,从而产生抗药性。该基因可作为抗药标记基因使用,建立稻瘟菌的基因转化系统。