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The pathway of sterol biosynthesis is highly conserved in all eucaryotic cells. We demonstrated structural and functional conservation of the rate-limiting enzyme of the mammalian pathway, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase), between the yeast Saccharomyces cerevisiae and humans. The amino acid sequence of the two yeast HMG-CoA reductase isozymes was deduced from DNA sequence analysis of the HMG1 and HMG2 genes. Extensive sequence similarity existed between the region of the mammalian enzyme encoding the active site and the corresponding region of the two yeast isozymes. Moreover, each of the yeast isozymes, like the mammalian enzyme, contained seven potential membrane-spanning domains in the NH2-terminal region of the protein. Expression of cDNA clones encoding either hamster or human HMG-CoA reductase rescued the viability of hmg1 hmg2 yeast cells lacking this enzyme. Thus, mammalian HMG-CoA reductase can provide sufficient catalytic function to replace both yeast isozymes in vivo. The availability of yeast cells whose growth depends on human HMG-CoA reductase may provide a microbial screen to identify new drugs that can modulate cholesterol biosynthesis.  相似文献   

3.
The amino acid sequence of the sodium channel alpha subunit from adult human skeletal muscle has been deduced by cross-species PCR-mediated cloning and sequencing of the cDNA. The protein consists of 1836 amino acid residues. The amino acid sequence shows 93% identity to the alpha subunit from rat adult skeletal muscle and 70% identity to the alpha subunit from other mammalian tissues. A 500 kb YAC clone containing the complete coding sequence and two overlapping lambda clones covering 68% of the cDNA were used to estimate the gene size at 35 kb. The YAC clone proved crucial for gene structure studies as the high conservation between ion channel genes made hybridization studies with total genomic DNA difficult. Our results provide valuable information for the study of periodic paralysis and paramyotonia congenita, two inherited neurological disorders which are caused by point mutations within this gene.  相似文献   

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Several cDNA clones for the mouse lactate dehydrogenase-X (LDH-X), a sperm-specific glycolytic enzyme, were isolated from mouse testicular cDNA libraries constructed in the bacteriophage vectors, lambda gt11 and gt10. The largest cDNA clone contains an insert of 1135 base pairs in length and an open reading frame that encodes a 332 amino acid polypeptide with a molecular weight of 35.89 kD. The deduced amino acid sequence of this protein is in close agreement with the published sequence of mouse LDH-X obtained by direct protein sequencing. Northern analysis of RNA isolated from different tissues detected a single size mRNA of 1.5 kilobases in mouse testis but not in brain or liver. The Ldh-x structural gene was estimated to be about 12 kb in size as demonstrated by Southern hybridization analysis of mouse genomic DNA using the full-length cDNA as a probe.  相似文献   

6.
We have developed a simple and versatile cDNA extension method using lambda-exonuclease-generated single-stranded DNA as a primer. This plasmid-based cDNA extension method can be used to synthesize unidirectional extensions of the existing cDNA clones or subcloned fragments of the untranslated and exon regions of genomic DNA clones. The method is simple to use and involves no addition of linkers or tailing. We have successfully used this method to isolate 4.6 kilobase pairs of chicken fatty acid synthase cDNA clones, starting from the fragment of a genomic clone coding for the untranslated region of the fatty acid synthase mRNA. About 2.8 kilobase pairs of the cDNA coding for the chicken fatty acid synthase has been sequenced. The sequence has an open reading frame coding for 945 amino acids of the fatty acid synthase. In the sequence, we have identified the enoyl reductase, NADPH binding region, a putative beta-ketoacyl reductase region, and the entire sequences of acyl carrier protein and the thioesterase domains. The arrangement of these partial activities in this sequence confirms the arrangement of these activities as determined through partial proteolytic mapping studies. The amino acid sequence of chicken fatty acid synthase deduced from cDNA sequences shows a high degree of homology with the rat fatty acid synthase sequence, suggesting that these multifunctional proteins are conserved evolutionarily.  相似文献   

7.
L Ji  M Becana  G Sarath    R V Klucas 《Plant physiology》1994,104(2):453-459
A cDNA encoding soybean (Glycine max [L.] Merr) ferric leghemoglobin reductase (FLbR), an enzyme that is postulated to play an important role in maintaining leghemoglobin in its functional ferrous state, has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full-length clone of FLbR cDNA was isolated by screening a lambda gt11 soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragment as a probe. The cDNA contained about 1.8 kb and had a coding sequence for 523 amino acids with a predicted molecular mass of 55,729 D, which included a putative 30-residue signal peptide and a 493-residue mature protein. Computer-aided analysis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20-50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reductase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypanothione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleotide-disulfide oxidoreductases from various organisms. Northern blot analysis using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater level of expression in nodules and leaves than in roots and stems. Southern blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome.  相似文献   

8.
The 'proliferating cell nuclear antigen' (PCNA), also known as cyclin, appears at the G1/S boundary in the cell cycle. Because of its possible relationship with cell proliferation, PCNA/cyclin has been receiving attention. PCNA/cyclin is a non-histone acidic nuclear protein with an apparent mol. wt of 33000-36000. The amino acid composition and the sequence of the first 25 amino acids of rabbit PCNA/cyclin are known. Using an oligonucleotide probe corresponding to the sequence of the first five amino acids, a cDNA clone for PCNA/cyclin was isolated from rat thymocyte cDNA library. The cDNA (1195 bases) contains an open reading frame of 813 nucleotides coding for 261 amino acids. The 3'-non-coding region is 312 nucleotides long and contains three putative polyadenylation signals. The mol. wt of rat PCNA/cyclin was calculated to be 28 748. The deduced amino acid sequence and composition of rat PCNA/cyclin are in excellent agreement with the published data. Using the cDNA probe, two species of mRNA (1.1 and 0.98 kb) were detected in rat thymocyte RNA. Southern blot analysis of total human genomic DNA suggests that there is a single gene coding for PCNA/cyclin. The deduced amino acid sequence of rat PCNA/cyclin has a similarity with that of herpes simplex virus type-1 DNA binding protein.  相似文献   

9.
We synthesized a DNA probe specific for the gene encoding eucaryotic DNA topoisomerase I by the polymerase chain reaction. The sequences of the primers for this reaction were deduced from the regions with extensive homology among the enzymes from the fission and budding yeasts, and the human. From the clones isolated by screening a Drosophila cDNA library with this DNA probe, two cDNA clones of 3.8 and 5.2 kb were characterized and completely sequenced. Both cDNA sequences contain an identical open reading frame for 972 amino acid residues. The 3.8 kb messenger RNA is likely generated by using a polyadenylation site 5' upstream to that used in generating the 5.2 kb mRNA. The predicted amino acid sequence shows that a segment of 420 amino acid residues at the amino terminus is hydrophilic, similar to the amino terminal 200 residues in the yeast and human enzymes. Furthermore, the Drosophila enzyme is unique in that the amino terminal 200 residues are enriched in serine and histidine residues; most of them are present in clusters. The rest of the Drosophila sequence is highly homologous to those from yeast and human enzymes. The evolutionarily conserved residues are identified and are likely the critical elements for the structure and function of this enzyme. A plasmid vector containing the cloned cDNA was constructed for the expression of Drosophila protein in Escherichia coli. The enzymatic and immunochemical analysis of the polypeptide produced in this heterologous expression system demonstrated that the expressed protein shares similar enzymatic properties and antigenic epitopes with DNA topoisomerase I purified from Drosophila embryos or tissue culture cells, thus establishing the bacterial expression system being useful for the future structure/function analysis of the Drosophila enzyme.  相似文献   

10.
Degenerate primers were designed based on all possible sequences of the N-terminal and C-terminal regions of Delonix regia trypsin inhibitor (DrTI). Five hundred sixty-one bp of polymerase chain reaction (PCR) product was amplified using the above degenerate primers and genomic DNA and cDNA of Delonix regia as a template. The amplified PCR products were cloned and sequenced. DNA sequence analysis of cDNA and genomic clones of DrTI have the same nucleotide sequence in the coding region, and manifested a genomic clone without intervening sequences in the coding region. The amino acid sequence deduced from the DrTI genomic and cDNA clones agreed with that identified via amino acid sequencing analysis, except that two amino acid residues, Ser and Lys, existed between residues Lys141 and Ser142. DrTI open reading frame was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in Escherichia coli to yield a glutathione S-transferase (GST)-fusion protein with a calculated molecular mass of about 45 kDa. The recombinant DrTI (reDrTI) was derived by treating the GST-DrTI fusion protein with thrombin. Both the reDrTI and GST-DrTI fusion protein exhibited a strong identical inhibitory effect on trypsin activity.  相似文献   

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