Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated full-length cDNA clones for apolipoprotein AI from a human fetal liver cDNA library made in the vector lambda gt10. One such clone, pBL13AI, was 965 nucleotides long and contained the entire preproapolipoprotein AI sequence, in addition to 86 nucleotides of 5' untranslated region and 75 nucleotides of the 3' untranslated region. The complete structure of this clone is presented. Furthermore, we have obtained a 20-kb genomic fragment from a human genomic library, encompassing the entire apo AI gene. Sequence analysis of the gene shows that the coding region is interrupted by three introns of 197, 187, and 588 bp, respectively. Digestion of the DNA of various individuals with the endonuclease Msp I revealed a new restriction site polymorphism in the apo AI gene.
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PMID:Isolation and DNA sequence of full-length cDNA and of the entire gene for human apolipoprotein AI--discovery of a new genetic polymorphism in the apo AI gene. 620 99

We report a Canadian kindred with a novel mutation in the apolipoprotein (apo) A-I gene causing analphalipoproteinemia. The 34-yr-old proband, product of a consanguineous marriage, had bilateral retinopathy, bilateral cataracts, spinocerebellar ataxia, and tendon xanthomata. High density lipoprotein cholesterol (HDL-C) was < 0.1 mM and apoA-I was undetectable. Genomic DNA sequencing of the proband's apoA-I gene identified a nonsense mutation at codon [-2], which we designate as Q[-2]X. This mutation causes a loss of endonuclease digestion sites for both BbvI and Fnu4HI. Genotyping identified four additional homozygotes, four heterozygotes, and two unaffected subjects among the first-degree relatives. Q[-2]X homozygosity causes a selective failure to produce any portion of mature apoA-I, resulting in very low plasma level of HDL. Heterozygosity results in approximately half-normal apoA-I and HDL. Gradient gel electrophoresis and differential electroimmunodiffusion assay revealed that the HDL particles of the homozygotes had peak Stokes diameter of 7.9 nm and contained apoA-II without apoA-I (Lp-AII). Heterozygotes had an additional fraction of HDL3-like particles. Two of the proband's affected sisters had documented premature coronary heart disease. This kindred, the third reported apoA-I gene mutation causing isolated complete apoA-I deficiency, appears to be at significantly increased risk for atherosclerosis.
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PMID:Apolipoprotein A-I Q[-2]X causing isolated apolipoprotein A-I deficiency in a family with analphalipoproteinemia. 828 91

In the process of screening apolipoprotein (apo) E genotypes in a population of subjects with lipid abnormalities, we have identified five subjects (one homozygote and four heterozygotes) with an abnormal 109 base pairs band following apo E restriction isotyping of amplified DNA with the restriction endonuclease CfoI. The polymerase chain reaction (PCR) products were cloned and their sequencing revealed a C-->A substitution at the first nucleotide of codon 136. This mutation resulted in an amino acid substitution Arg to Ser, previously described as apo E2 Christchurch. Family studies were carried out for four of the probands. In these kindreds, stepwise multiple regression analyses indicated that 78% of the cholesterol variability in men was predicted by body mass index, age and the rare apo E2 (Arg136-->Ser) variant. In women, age and the apo E2 (Arg136-->Ser variant predicted 54.9% of the variability in cholesterol levels. Linkage analysis suggested that the presence of the apo E2 (Arg136-->Ser) variant was linked with the occurrence of cholesterol enriched triglyceride rich lipoproteins and with an incomplete dominance of type III hyperlipoproteinemia. Our data indicates that this mutation may be a relatively common cause of dyslipidemia in the Spanish population.
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PMID:Incomplete dominance of type III hyperlipoproteinemia is associated with the rare apolipoprotein E2 (Arg136-->Ser) variant in multigenerational pedigree studies. 872 10

The ribonuclease protection assay procedure described enables the relative quantitation of either single mRNAs or multiple mRNA species simultaneously in a sample of total RNA and demonstrates its applicability to two systems of relevance to the study of apolipoproteins, namely, liver tissue and liver-derived cell lines in culture. The main requirements of the method are the availability of cDNA cloned into a vector that directs the transcription of antisense RNA for the preparation of radioactive probes, and choice of suitable restriction endonuclease sites for linearizing the cDNA so that the final protected products of the various mRNA species are sufficiently different in size to allow their separation. For moderately abundant apolipoprotein mRNAs in rat liver, the method is sensitive down to 1 microgram total RNA. Other experimental sources of RNA or the assay of less abundant mRNA species may require a larger amount of starting material. These aspects of the assay need to be determined for each probe/ tissue system to be studied. The need for adjustments to the specific radioactivity of the probes will depend on the relative abundance of the target mRNA molecules and can be readily determined empirically. The rationale for varying the specific radioactivities to facilitate multiple assays as suggested here is both simple and effective. A preliminary assay provides information on the relative levels of the mRNA species of interest, and the step of preparing, in parallel, a sample of unlabeled antisense RNA provides the means for quantitative dilution of specific radioactivity of the 32P-labeled RNA probe where required.
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PMID:Determination of apolipoprotein mRNA levels by ribonuclease protection assay. 874 22

Estrogen-mediated accumulation of the avian apolipoprotein (apo) II mRNA is in part due to its stabilization. To identify the biochemical activity responsible for this effect, radiolabeled, capped, and polyadenylated apoII mRNA was incubated in vitro in liver cytosolic extracts from roosters who received either estrogen (estrogen-treated extract) or the vehicle (control extract) parenterally. The mRNA was very stable in estrogen-treated extract but was rapidly degraded in control extract. The RNA was degraded predominantly by endonuclease rather than exonuclease activity. The addition of the estrogen-treated extract to the control extract prevented the degradation of the mRNA in trans. This biochemical activity was heat labile and was also destroyed by proteinase K but not by micrococcal nuclease, indicating that estrogen treatment resulted in the expression of a protein in the liver that stabilized the apoII mRNA by inhibiting its nucleolytic degradation. This mRNA stabilization factor was labile around 60 degrees C, whereas the RNase remained stable up to 80 degrees C. Studies on mRNA protein interaction showed that both control and estrogen-treated extracts contain mRNA-binding (mRNP) proteins that bind apoII mRNA. An increased binding to apoII mRNA by a subset of these proteins was observed with estrogen-treated extract as compared with the control extract. This activity, although it afforded complete protection from nucleolytic degradation to apoII and apo A1 mRNAs, appeared to provide less protection to mRNAs encoding chicken serum albumin and vitellogenin, suggesting differential stabilization of mRNAs. These studies indicate that a cytosolic mRNA-stabilization factor, providing apoII mRNA complete protection from nucleolytic degradation, is expressed in the avian liver upon estrogen treatment. This appears to be the first time that a biochemical activity responsible for hormone-mediated stabilization of mRNAs and estrogen induction of mRNA binding by specific mRNPs have been identified and partially characterized in vitro.
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PMID:In vitro characterization of an estrogen-regulated mRNA stabilizing activity in the avian liver. 877 38

This study addresses two important technical problems: how to perform targeted alterations such as site-directed mutagenesis and deletions in large fragments of DNA and how to construct full-length genes from two partly overlapping bacterial artificial chromosome (BAC) plasmids. Given the size and the lack of convenient unique restriction sites in these large-insert bacterial clones, these are nontrivial tasks. Here we describe a simple and efficient protocol based on RecA-assisted restriction endonuclease (RARE) cleavage, a method that enables sequence-specific cleavage of genomic DNA. The same protocol has been used with minor modifications to introduce site-specific mutations into an apolipoprotein-B 90-kb P1 clone, to generate deletions in a 160-kb BAC, and to generate a 160-kb BAC containing the complete 92-kb gene for low-density lipoprotein-related protein-1 (LRP-1) from two smaller overlapping BACs ("BAC marriage").
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PMID:A simple and efficient method for making site-directed mutants, deletions, and fusions of large DNA such as P1 and BAC clones. 893 37

Several environmental and genetic factors are associated with high levels of cholesterol. Hypercholesterolemia is the main phenotype of Familial Defective Apolipoprotein B and Familial Hypercholesterolemia that are caused by mutations at the apolipoprotein (apo) B and LDL receptor genes, respectively. Identification of the specific genetic alteration associated with hypercholesterolemia is an important issue in clinical diagnosis of high risk for CAD. Apo B gene mutations and polymorphisms are usually screened by SSCP, DGGE, and heteroduplex, which must be confirmed by DNA sequencing or by direct detection using PCR techniques. In this study, we have optimized a PCR-RFLP procedure for identification of 3500Q and 3531 mutations and MspI polymorphism at the apo B gene. The technique can be performed in a single reaction, using the restriction endonuclease MspI for simultaneous detection of 3500Q mutation and MspI polymorphism, and NsiI for detection of 3531 mutation. The procedure was validated by analysis of control DNA samples from individuals carrying these mutations. Screening of 186 Brazilian hypercholesterolemic individuals showed that the frequency of the M-allele (7.8%) of MspI polymorphism was similar to that found in other individuals with CAD. However, neither 3500Q nor 3531 mutations were detected in this group. In conclusion, this procedure is simple and rapid, being easily introduced in clinical laboratories for direct detection of the more frequent mutations at the apo B gene associated with hypercholesterolemia.
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PMID:Rapid detection of 3500Q and 3531 mutations and MspI polymorphism in exon 26 at the apolipoprotein B gene. 1117 Feb 32

The role of apolipoprotein E (apoE) genotypes in modulating plasma lipid and apolipoprotein levels was studied in 112 patients with Type 2 diabetes mellitus (T2DM) and 94 healthy individuals. ApoE genotypes were identified by PCR amplification and subsequent restriction endonuclease digestion. The apoE allele and genotype frequencies were similar in both the diabetic and control subjects. The apoE allele frequencies were found to be 74.3 for e3, 10.1 for e2, 15.6 for e4 in the diabetic group, and 68.1 for e3, 13.2 for e2 and 18.7 for e4 in the control group. Sex-specific genotypic distribution of apoE polymorphism did not differ between the study groups. To elucidate the association of apoE with lipid abnormalities with respect to gender, serum lipid and apolipoprotein levels were compared among apo e2 (e2/2 and e3/2), e3 (e3/3) and e4 (e4/3 and e4/4) groups of T2DM and control subjects. Apo e2 allele was found to be associated to triglycerides for both sexes, and associated to glucose, and BMI only in females. Subjects with e2 allele had higher levels of BMI, glucose and triglyceride in comparison to e3 and e4. Our data suggest that genetic variation at the apoE locus in Turkish subjects is a genetic factor that influences lipid levels. Further studies attempting to correlate apoE polymorphism with lipid profile in a large number of individuals would be helpful in establishing the true significance of this polymorphism in the Turkish population.
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PMID:Apolipoprotein E polymorphism in Turkish subjects with Type 2 diabetes mellitus: allele frequency and relation to serum lipid concentrations. 1629 48


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