Analysis of apolipoprotein E7 (apolipoprotein E-Suita) gene from a patient with hyperlipoproteinemia

We have isolated and analyzed apolipoprotein E7 gene from a patient with hyperlipoproteinemia. Apolipoprotein E7 (apolipoprotein E-Suita) is a variant of apolipoprotein E with four additional units of positive charge compared to apolipoprotein E3, which is the major isoform of apolipoprotein E. The heterozygous gene of apolipoprotein E7/3 from the patient was cloned into lambda phage. The cloned apolipoprotein E genes were subcloned into a murine retrovirus shuttle vector and were expressed. Two out of five clones expressed apolipoprotein E7. The analysis of the nucleotide sequence of the exon and exon-intron boundary regions has shown two G-to-A nucleotide substitutions in the 548 and 551 nucleotide positions from the 5'-end of the fourth exon. These two base substitutions change the amino acid residues -Glu-Glu- to -Lys-Lys- at the 244 and 245 positions from the amino-terminus of the mature protein, and give four additional units of positive charge to the molecule.     

Apolipoprotein E-4Philadelphia (Glu13----Lys,Arg145----Cys). Homozygosity for two rare point mutations in the apolipoprotein E gene combined with severe type III hyperlipoproteinemia.

The molecular defect in a 24-year-old white female with severe type III hyperlipoproteinemia has been elucidated. The patient's apolipoprotein (apo) E migrated in the apoE-4 position on isoelectric focusing gels. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the apoE-4 variant had a smaller apparent molecular weight than apoE-4(Cys112----Arg). Sequence analysis of DNA amplified with the polymerase chain reaction revealed two nucleotide substitutions in the proband's apoE gene. A C to T mutation converted arginine (CGT) at position 145 of the mature protein to cysteine (TGT) thus creating the apoE-2 variant. A second G to A substitution at amino acid 13 led to the exchange of lysine (AAG) for glutamic acid (GAG), thereby adding 2 positive charge units to the protein and producing the apoE-5 variant. Computer analysis of the apoE-4Philadelphia gene revealed that the G to A mutation in exon 3 resulted in the loss of an AvaI restriction enzyme site. The second mutation, a C to T substitution in the fourth exon of the apoE gene, eliminated a cleavage site for the enzyme BbvI. Using these restriction fragment length polymorphisms as well as DNA sequence analysis we have demonstrated that the patient is homozygous for both point mutations in the apoE gene.     

Structure and expression of mouse apolipoprotein E gene

The mouse apolipoprotein E gene was isolated from a genomic library by screening with a cDNA probe. DNA including apolipoprotein E gene plus segments 2.5 kilobases upstream and 0.3 kilobase downstream of the coding region was transfected into NIH3T3 cells. The cells expressed the same-size apolipoprotein E mRNA and protein as those produced by mouse endogenously. The nucleotide sequence of the gene plus 5' and 3' flanking regions (one kilobase each) was determined. The sequence of the mouse apoliprotein E gene was highly homologous to that of the rat gene, not only in the coding regions but also in the non-coding and intron regions. The mouse and the human apolipoprotein E genes were homologous in the 5' proximal flanking region up to about 200 nucleotides as well as in the four exons. This proximal region was highly conserved for the genes of mouse, rat and human; the relative positions of the "TATA box" and the two copies of "GC box" were identical.     

Cloning and nucleotide sequence of the aroA gene of Bordetella pertussis.

The aroA locus of Bordetella pertussis, encoding 5-enolpyruvylshikimate 3-phosphate synthase, has been cloned into Escherichia coli by using a cosmid vector. The gene is expressed in E. coli and complemented an E. coli aroA mutant. The nucleotide sequence of the B. pertussis aroA gene was determined and contains an open reading frame encoding 442 amino acids, with a calculated molecular weight for 5-enolpyruvylshikimate 3-phosphate synthase of 46,688. The amino acid sequence derived from the nucleotide sequence shows homology with the published amino acid sequences of aroA gene products of other microorganisms.     

Evolution of the apolipoproteins. Structure of the rat apo-A-IV gene and its relationship to the human genes for apo-A-I, C-III, and E

We have determined the nucleotide sequence of the rat apolipoprotein (apo-) A-IV gene and analyzed its structural and evolutionary relationships to the human apolipoprotein A-I, E, and C-III genes. The rat A-IV gene is 2.4 kilobases in size and consists of three exons (142, 126, and 1157 base pairs) interrupted by two introns (277 and 673 base pairs). The 5'-nontranslated region and most of the signal peptide are encoded by the first exon. Thus, the apo-A-IV gene lacks an intron in the 5'-nontranslated region of its mRNA in contrast to all other known apolipoprotein genes. Sequences coding for amphipathic docosapeptides span both the second and third exons of the rat A-IV gene. We demonstrate that this is also true for the human apolipoprotein genes. This gene family seems to have evolved by the duplication of an ancestral minigene that resulted in the formation of two exons. Thereafter, evolution of these sequences was dominated by intraexonic amplification of repeating units coding for amphipathic peptides. Sequence divergence of these repeats resulted in the functional differentiation of the apolipoproteins. However, conservation of the fundamental amphipathic pattern allowed members of this protein family to retain their lipid-binding properties.     

Human adenylate kinase deficiency associated with hemolytic anemia. A single base substitution affecting solubility and catalytic activity of the cytosolic adenylate kinase

Adenylate kinase deficiency in the erythrocyte is a rare genetic disorder associated with hemolytic anemia. To determine the molecular basis of this disorder, we first cloned the normal gene encoding human cytosolic adenylate kinase (AK1) and determined the structure. The gene was 12 kilobase pairs long and was split into 7 exons. The structures of 5'- and 3'-flanking regions were determined by primer extension and RNA blot analysis. The results showed that two species of mRNA with 0.9 and 2.5 kilobases, which differed at the 3'-end portion, were generated by the AK1 gene. Alu sequences were found in the largest intron (intron 5) and in the noncoding region of exon 7. Next, both alleles of the AK1 gene were cloned from DNA of a patient bearing the adenylate kinase deficiency and their nucleotide sequences determined. A transition (C----T) was found in exon 6 on an allele, which resulted in an Arg to Trp (CGG----TGG) substitution at the 128th residue of AK1. Since chicken AK1 is highly homologous to human AK1 with respect to the amino acid sequence, we introduced an Arg to Trp substitution to chicken AK1 at the same position by oligodeoxynucleotide-directed mutagenesis. The mutant chicken AK1 expressed in Escherichia coli showed a reduced catalytic activity as well as a decreased solubility and a change in affinity to phosphocellulose. Thus it was considered that the observed C----T transition was a cause of the decreased AK1 activity of the patient's erythrocyte. Analysis on phosphocellulose chromatography of erythrocyte AK1 of the patient and parents revealed that the patient's mutant allele was derived from the mother.     

小肽多拷贝基因表达载体的构建及其高效表达(英文)

介绍一种快速、高效构建小肽多拷贝基因表达载体的策略 ,并构建了相应的表达载体pETE coT .用人工合成的编码 2 8个氨基酸残基的胸腺素α1基因为模型 ,采用限制酶EcoT14I识别序列CCAAGG为小肽基因两末端序列 ,利用其酶切后可产生非镜相对称粘性末端 ,一次连接反应就构建出一系列不同基因拷贝数的表达载体 ;在小肽基因两端分别引进编码FactorXa和羟胺蛋白切割位点的序列 ,表达出的融合蛋白可被FactorXa和羟胺剪切出不残留任何外源氨基酸的小肽 .不同拷贝数的小肽融合蛋白在大肠杆菌BL2 1(DE3)中均获得高效表达 .     

Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA.

A biologically active molecular clone of BALB/Moloney mink cell focus-forming (Mo-MCF) proviral DNA has been reconstructed in vitro. It contains the 5' half of BALB/Moloney murine leukemia virus (Mo-MuLV) DNA and the 3' half of BALB/Mo-MCF DNA. The complete nucleotide sequence of the env gene and the 3' long terminal repeat (LTR) of the cloned Mo-MCF DNA has been determined and compared with the sequence of the corresponding region of parental Mo-MuLV DNA. The substitution in the Mo-MCF DNA encompasses 1,159 base pairs, beginning in the carboxyl terminus of the pol gene and extending to the middle of the env gene. The Mo-MCF env gene product is predicted to be 29 amino acids shorter than the parental Mo-MuLV env gene product. The portion of the env gene encoding the p15E peptide is identical in both viral DNAs. There is an additional A residue in the Mo-MCF viral DNA in a region just preceding the 3' LTR. The nucleotide sequence of the 3' LTR of Mo-MCF DNA is similar to that of the 5' LTR of BALB/Mo-MuLV DNA with the exception of two single base substitutions. We conclude that the sequence substitution in the env gene is responsible for the dual-tropic properties of Mo-MCF viruses.     

Drosophila melanogaster genes for U1 snRNA variants and their expression during development.

We have cloned and characterized a complete set of seven U1-related sequences from Drosophila melanogaster. These sequences are located at the three cytogenetic loci 21D, 82E, and 95C. Three of these sequences have been previously studied: one U1 gene at 21D which encodes the prototype U1 sequence (U1a), one U1 gene at 82E which encodes a U1 variant with a single nucleotide substitution (U1b), and a pseudogene at 82E. The four previously uncharacterized genes are another U1b gene at 82E, two additional U1a genes at 95C, and a U1 gene at 95C which encodes a new variant (U1c) with a distinct single nucleotide change relative to U1a. Three blocks of 5' flanking sequence similarity are common to all six full length genes. Using specific primer extension assays, we have observed that the U1b RNA is expressed in Drosophila Kc cells and is associated with snRNP proteins, suggesting that the U1b-containing snRNP particles are able to participate in the process of pre-mRNA splicing. We have also examined the expression throughout Drosophila development of the two U1 variants relative to the prototype sequence. The U1c variant is undetectable by our methods, while the U1b variant exhibits a primarily embryonic pattern reminiscent of the expression of certain U1 variants in sea urchin, Xenopus, and mouse.     

Molecular cloning and nucleotide sequence of a pectin lyase gene from Pseudomonas marginalis N6301.

A pectin lyase (PNL;EC4.2.2.10) gene of Pseudomonas marginalis N6301 was cloned and expressed in Escherichia coli. We purified PNL from P. marginalis N6301 and determined N-terminal 33 amino acids sequence. From this sequence, we synthesized two oligonucleotide probes. From the analysis of Southern hybridization, 2. 1kb EcoRI-SmaI fragment from the chromosomal DNA of P. marginalis was found to hybridize with oligonucleotide probes. Then, we cloned the fragment into pUC119 vector and transformed into E. coli DH5 alpha. A plasmid thus obtained was designated as pPNL6301. E. coli DH5 alpha harboring pPNL6301 expressed PNL activity. The nucleotide sequence of pn1 gene in the plasmid pPNL6301 encoding PNL from P. marginalis N6301 was determined. The structural gene of pn1 consisted of 936 base pairs. An open reading frame that encodes a 34,103 dalton polypeptide composed of 312 amino acids was assigned. The molecular weight of the polypeptide predicted from the amino acid composition was close to that of PNL of P. marginalis N6301 determined. The nucleotide sequence of the 5'-flanking region of pn1 gene showed the presence of the consensus sequence of LexA binding site, Pribnow box and ribosome binding site as found in Escherichia coli. The amino acid sequence homology of PNLs and nucleotide sequence homology of pn1 gene between P. marginalis N6301 and E. carotovora Er were 60.8% and 57.2%, respectively.     

Human apolipoprotein E mRNA. cDNA cloning and nucleotide sequencing of a new variant

The complete nucleotide sequences of three cloned cDNAs corresponding to human liver apolipoprotein E (apo-E) mRNA were determined. Analysis of the longest cDNA showed that it contained 1157 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 61 nucleotides, a signal peptide region corresponding to 18 amino acids, a mature protein region corresponding to 299 amino acids, and a 3'-terminal nontranslated region of 142 nucleotides. The inferred amino acid sequences from two cDNAs were identical and corresponded to the amino acid sequence for plasma apo-E3 that has been reported previously ( Rall , S. C., Jr., Weisgraber , K. H., and Mahley , R. W. (1982) J. Biol. Chem. 257, 4171-4178). The third cDNA differed from the other two cDNAs in five nucleotide positions. Three of these differences occurred in the third nucleotide position of amino acid codons, resulting in no change in the corresponding amino acids at residues Val-85, Ser-223, and Gln-248. The other two altered nucleotides occurred in the first nucleotide position of codons, leading to changes in the amino acids encoded. In the variant sequence, a threonine replaced the normal alanine at residue 99 and a proline replaced the normal alanine at residue 152. We have concluded that the human liver donor was heterozygous for the epsilon 3 genotype. The variant cDNA corresponds to a new, previously undescribed variant form of apo-E in which the amino acid substitutions of the protein are electrophoretically silent; it would probably be undetectable by standard apo-E phenotyping methods. The amino acid substitution at position 152 occurs in a region of apo-E that appears to be important for receptor binding, and it may have clinical significance.     

Duplication followed by deletion accounts for the structure of an Indian deletion beta (0)-thalassemia gene.

Nucleotide sequence analysis of a cloned deletion beta-globin gene from a patient with beta(0)-thalassemia demonstrates a 619 nucleotide deletion extending from the 3' third of the second intervening sequence through 209 bases of 3' flanking DNA. However, an additional novel heptanucleotide was identified between the deletion endpoints, suggesting a complex etiology for this rearrangement.     

Nucleotide sequences of genes encoding heat-stable and heat-labile glyceraldehyde-3-phosphate dehydrogenases; amino acid sequence and protein thermostability

The structural gene (gapST) encoding glyceraldehyde-3-phosphate dehydrogenase (GPDH; EC 1.2.1.12) from Bacillus stearothermophilus has been cloned in Escherichia coli using plasmid pBR322 as a vector; the homologous gene (gapCO) from Bacillus coagulans was cloned from a phage lambda library. Expression of the cloned gap genes revealed that, as in the wild-type (wt) organisms, the GPDH from B. stearothermophilus (GPDH-ST) was intrinsically heat stable (hs) and that from B. coagulans (GPDH-CO) heat labile (hl). The cloned gap genes were sequenced and the deduced amino acid (aa) sequences were found to be highly conserved (91.6% homology), despite the large difference in thermostability between these two enzymes. Of the 28 aa which differ between the two proteins, most of which occur in the middle third of the monomeric subunit, 5 aa involve replacement of alanine in the hl GPDH-CO, by proline in the hs GPDH-ST, and are especially interesting in terms of their potential contributions to thermostability. Conservation at the DNA level is equally dramatic, with the two gap genes exhibiting 93.3% nucleotide sequence homology. These highly expressed genes exhibit an equivalent codon bias, which more closely resembles that of highly expressed E. coli genes, than that of B. stearothermophilus genes whether highly or weakly expressed.     

A fusion gene in man: DNA sequence analysis of the abnormal globin gene of Hemoglobin Miyada

An abnormal globin gene from a patient heterozygous for Hemoglobin Miyada was cloned and sequenced. The results indicated that the 5′ flanking region and the 5′ side of the gene were identical to those of a β-globin gene and that the 3′ side was identical to that of a γ-globin gene. The part of the gene identical to a β-globin gene shifted to the part identical to the δ-globin gene somewhere in a homologous sequence region between the third nucleotide of the 17th codon and the second nucleotide of the 22nd codon of these two genes. Thus, results of analysis of the nucleotide sequence support the idea that the abnormal globin gene of Hemoglobin Miyada was generated as a fusion gene by unequal crossing over between a β- and a δ-globin gene.     

Apolipoprotein E1 Lys-146----Glu with type III hyperlipoproteinemia.

During the screening of samples obtained from 5 individuals with type III hyperlipidemia, we identified a variant of apolipoprotein (apo) E which exhibited a discrepancy in apo E phenotype showing the E3/E1 isoform on isoelectric focusing (IEF) analysis and E3/E3 on gene analysis. Sequence analysis of the DNA of the proband that was amplified by PCR and subcloned, revealed a single substitution of one lysine (AAG) for one glutamic acid (GAG) at position 146, thereby adding two negatively charged units to apo E3. This defect had been described only for apo E1 to date (Mann et al. (1989) Clin. Res. 37, 520A (abstract)). In this case, PCR-mediated site-directed mutagenesis was used to identify the structural alterations forming the abnormal E1 genotype in the proband's family. Purified apo E1 Lys-146----Glu showed less than 10% of binding activity to apo B, E receptor on human skin fibroblasts compared with apo E3. This substitution demonstrates that Lys-146 is essential for the binding of apo E to the receptor.     

Developmental changes in the pattern of larval beta-globin gene expression in Xenopus laevis. Identification of two early larval beta-globin mRNA sequences

We have analysed beta-globin mRNA sequences in total RNA extracted from embryos and tadpoles of Xenopus laevis at different stages of development and we have identified the most abundantly transcribed beta-globin mRNA (beta T1). The entire nucleotide sequence of a cDNA clone corresponding to this mRNA is known. We have now identified the gene corresponding to this mRNA and we have determined the nucleotide sequences of its immediate 5'-flanking region. Using a DNA fragment from within the coding region of the cloned beta T1 cDNA we show, by primer extension analysis, that beta T1 mRNA is first detectable at stage 28-32 of development. This is the time at which the first presumptive erythropoietic tissue, the ventral blood island, becomes observable histologically. We show that two minor beta-globin genes, distinct from beta T1, are expressed during early stages of development, and that their expression ceases shortly after the beginning of the feeding stage. We term these two early larval genes beta E1 and beta E2. A third minor beta-globin gene is expressed during early development but, unlike beta E1 and beta E2, it is also expressed throughout subsequent larval development. We term this gene beta T2 and show that it corresponds to a gene previously termed beta LII. Finally, using a primer derived from the major adult beta-globin gene (beta 1), we have analysed the accumulation of the major adult beta-globin mRNA during larval development, and we show that this sequence does not accumulate to any significant level before metamorphosis.     

The molecular basis of a familial apoE deficiency. An acceptor splice site mutation in the third intron of the deficient apoE gene

The molecular basis of the familial apoE deficiency was investigated by gene cloning and comparative expression studies of the normal and the deficient apoE gene. For the latter studies the apoE genes were placed under the control of the mouse metallothionein I promoter in a bovine papilloma virus vector. The studies showed that in the normal gene the mouse metallothionein I promoter directs the synthesis of normal apoE mRNA and protein. In contrast, in the deficient apoE gene the same promoter directs the synthesis of two abnormal apoE mRNA forms, which are similar to those observed in the peripheral blood monocyte macrophage cultures of the patient. Restriction analysis of the cloned gene and partial DNA sequence has shown an A to G substitution in the penultimate 3' nucleotide of the third intron of the apoE gene. This single base substitution abolishes the correct 3' splice site thus creating two abnormally spliced mRNA forms. The smaller form contains 53 nucleotides and the larger form contains the entire third intron of the apoE gene. Both of these mRNA species contain chain termination codons within the intronic sequence and code for short apoE peptides that are not detectable by gel electrophoretic techniques. These findings show that this form of familial apoE deficiency results from a point mutation in the 3' splice junction of the third intron of the apoE gene. Furthermore, the expression system we have employed to study apoE deficiency can be utilized to analyze a broad spectrum of genetic diseases associated with mRNA processing mutations.