Biomedical subjects

S K Karathanasis

Publications and source records attributed to S K Karathanasis.

52 records · Page 3Linked to original sources

PubMed1985

Isolation, characterization, and mapping to chromosome 19 of the human apolipoprotein E gene.

The human apo-E gene has been isolated from a lambda phage library using as a probe the previously reported apo-E cDNA clone pE-301. Lambda apo-E was mapped and subcloned, and the apo-E gene was completely sequenced. The DNA sequence was compared with that of a near full length cDNA clone pE-368 and revealed three introns. The first intron was in the region that corresponds to the 5' untranslated region of apo-E mRNA. The second intron interrupted the codon specifying amino acid -4 of the apo-E signal peptide. The third intron interrupted the codon specifying amino acid 61 of the mature protein. Analysis of the DNA sequence revealed four Alu sequences. Two were in opposite orientations in the second intron, and one each occurred in the regions 5' and 3' to the apo-E gene. There were two base differences between the apo-E gene sequence and the sequence derived from the cDNA clones. At the codon for amino acid residue 112, the apo-E gene contained CGC, specifying Arg, whereas the cDNA contained TGC, specifying Cys. The other base difference was in the area corresponding to the 5' untranslated region of apo-E mRNA. Apo-E is commonly polymorphic in the population and the data suggest that the genomic clone was derived from the epsilon 4 apo-E allele, whereas the cDNA clones were derived from the epsilon 3 apo-E allele. S1 nuclease protection and primer extension experiments allowed the tentative assignment of the cap site of apo-E mRNA to the A approximately 44 base pairs upstream of the GT that begins the first intron. The sequence TATAATT was identified beginning 33 base pairs upstream of the proposed cap site and is presumably one element of the apo-E promoter. Finally, the apo-E gene was mapped in the human genome to chromosome 19 through the use of DNA probes and human-rodent somatic cell hybrids.

Apolipoprotein A-I↗

PubMed1985

Apolipoprotein multigene family: tandem organization of human apolipoprotein AI, CIII, and AIV genes.

The genes for two of the proteins of the plasma lipid transport system, apolipoprotein AI (apoAI) and CIII (apoCIII) are closely linked in the human genome. An approximately equal to 30-kilobase (kb) DNA segment containing these genes and their flanking sequences has been cloned and extensively characterized. Hybridization studies revealed that a DNA fragment located 12 kb 3' to the apoAI gene contains sequences homologous to a 1.8-kb mRNA transcript in human fetal intestine and adult liver but not in fetal liver, kidney, heart, brain, or muscle. This DNA fragment was used as a probe to isolate a clone from an adult human liver cDNA library. The nucleotide sequence of this clone is 74.8% homologous to the cDNA sequence of rat apolipoprotein AIV (apoAIV), another protein of the lipid transport system, and codes for a protein that is 58.6% identical to rat apoAIV. These results indicate that apoAI, apoCIII, and apoAIV genes are closely linked in the human genome and suggest that all three of them are derived from a common ancestral precursor.

Amino Acid Sequence↗

PubMed1985

RNA-mediated gene duplication: the rat preproinsulin I gene is a functional retroposon.

Rats and mice have two, equally expressed, nonallelic genes encoding preproinsulin (genes I and II). Cytological hybridization with metaphase chromosomes indicated that both genes reside on rat chromosome I but are approximately 100,000 kilobases apart. In mice the two genes reside on two different chromosomes. DNA sequence comparisons of the gene-flanking regions in rats and mice indicated that the preproinsulin gene I has lost one of the two introns present in gene II, is flanked by a long (41-base) direct repeat, and has a remnant of a polydeoxyadenylate acid tract preceding the downstream direct repeat. These structural features indicated that gene I was generated by an RNA-mediated duplication-transposition event involving a transcript of gene II which was initiated upstream from the normal capping site. Sequence divergence analysis indicated that the pair of the original gene and its retroposed, but functional, counterpart (which appeared about 35 million years ago) is maintained by strong negative selection operating primarily on the segments encoding the chains of the mature hormone, whereas the segments encoding the parts of the polypeptide that are eliminated during processing and also the introns and the flanking regions are evolving neutrally.

Animals↗

PubMed1985

Isolation and characterization of cDNA clones corresponding to two different human apoC-III alleles.

We have recently reported that the human apolipoprotein A-I (apoA-I) and apolipoprotein C-III (apoC-III) genes are physically linked and that the presence of a DNA insertion in the apoA-I gene is correlated with apoA-I-apoC-III deficiency in patients with premature atherosclerosis. In addition, the presence of a polymorphic restriction endonuclease site (SacI) in the 3' noncoding region of apoC-III mRNA has been correlated with hypertriglyceridemia in humans. In this study, we report the isolation and characterization of cDNA clones containing the entire apoC-III mRNA coding sequence. The nucleotide-derived apoC-III amino acid sequence indicates that the apoC-III primary translational product contains a 20 amino acid N-terminal extension, which conforms with the general properties of known signal peptides, and is highly homologous to the recently reported rat apoC-III signal peptide. The DNA-derived apoC-III amino acid sequence differs from the previously reported apoC-III amino acid sequence at four amino acid residues. More specifically, at positions +32, +33, +37, +39, the DNA sequence predicts Glu, Ser, Gln, Ala, respectively, while the previously reported sequence specifies Ser, Gln, Ala, Gln, respectively. Finally, isolation and characterization of apoC-III cDNA clones, with or without the polymorphic SacI restriction site, indicated that the apoC-III nucleotide sequence corresponding to the Sac+ and Sac- clones differs at three nucleotide sites; however, the amino acid sequence specified by the Sac+ and Sac- alleles is identical.

Alleles↗

PubMed1984

Synthesis, intracellular processing, and signal peptide of human apolipoprotein E.

Northern blotting analysis has shown apo-E mRNA synthesis by human liver, HepG2 cells, and primary cultures of human monocyte macrophages but not by the macrophage-like cell line U937 and normal or transformed human fibroblasts. Cell-free translation has shown that the primary translation product of apo-E consists of one major and one minor isoprotein of apparent Mr = 28,500 and isoelectric points 6.20 and 6.02, respectively. These isoproteins differ by +1 and 0 charges from apo-E3 and have been designated preapo-E. Co-translational treatment of mRNA with dog pancreatic membranes converts both preapo-E isoproteins to a form which is undistinguishable by two-dimensional gel electrophoresis from plasma apo-E3. The isolation and nucleotide sequence analysis of a full length apo-E cDNA clone has shown that preapo-E contains an 18-amino acid NH2-terminal signal peptide compared to plasma apo-E. The signal peptide sequence is: MetLysValLeuTrpAlaAlaLeuLeuValThrPheLeuAlaGlyCysGlnAla. Comparison of co-translationally modified apo-E with intracellular, secreted, and plasma forms indicates that after the intracellular cleavage of the signal peptide, the protein is glycosylated with carbohydrate chains containing sialic acid, secreted as sialoapo-E (apo-Es), and subsequently desialated in plasma. These findings demonstrate that apo-E is synthesized as preprotein and undergoes intracellular proteolysis and glycosylation and extracellular desialation to attain the major asialoapo-E isoprotein form observed in plasma.

Amino Acid Sequence↗

PubMed1984

Guinea pig preproinsulin gene: an evolutionary compromise?

We characterized a clone carrying the guinea pig preproinsulin gene, which, in contrast to other mammalian preproinsulin genes, is highly divergent in its regions encoding the B and A chains of mature insulin. Blot hybridization analysis indicates that this gene is present in only one copy in the guinea pig genome and that other normal or mutated preproinsulin genes do not exist in this animal. Moreover, the position of introns in this gene and the homology of its 3' flanking region to the corresponding regions of other sequenced mammalian genes show that it has been derived from the common mammalian stock. The rapid evolution of the region encoding the B and A chains can be interpreted, according to our sequence-divergence analysis, as due to the fixation of both neutral and adaptive mutations.

Amino Acid Sequence↗

PubMed1983

An inherited polymorphism in the human apolipoprotein A-I gene locus related to the development of atherosclerosis.

Epidemiological studies have identified elevated low density lipoprotein (LDL) and diminished high density lipoprotein (HDL) cholesterol levels as risk factors for coronary artery disease. The major protein component of HDL is apoprotein A-I (apo A-I), a polypeptide of 243 amino acids of known primary amino acid sequence. This apoprotein serves as a cofactor for the plasma lecithin-cholesterol acyltransferase (LCAT) enzyme responsible for the formation of most cholesteryl esters in plasma, and also promotes cholesterol efflux from cells. The primary translation product of apo A-I contains both a pre and a pro segment, and post-translational processing of apo A-I may be involved in the formation of the functional plasma apo A-I isoproteins. Defective apo A-I processing may be the underlying problem in Tangier disease, in which patients have low plasma HDL and apo A-I levels despite normal apo A-I synthesis. Patients have been reported with conditions distinct from Tangier disease in whom severe deficiency or absence of apo A-I has been associated with very low HDL levels and severe coronary artery disease. We have now examined the apo A-I gene in two such patients and their first degree relatives. These patients have been reported to have skin and tendon xanthomas, corneal clouding and severe premature coronary atherosclerosis associated with very low HDL levels and deficiencies of two apoproteins, apo A-I and apo C-III. We show that both probands are homozygous for a defect in the apo A-I gene locus.

Apolipoprotein A-I↗

PubMed1983

Isolation and characterization of the human apolipoprotein A-I gene.

We have recently shown that an inherited polymorphism occurring in the human apolipoprotein A-I (apo A-I) gene is related to decreased high density lipoprotein and apo A-I levels in the plasma of two patients with severe premature atherosclerosis. Analysis of the molecular basis of this polymorphism and its possible effects on apo A-I gene expression requires direct comparison of both normal and polymorphic apo A-I alleles. Here we report the isolation and characterization of the normal human apo A-I gene and we show that the gene is interrupted by three intervening sequences, IVS-1, IVS-2, and IVS-3, occurring in the 5' noncoding region of apo A-I mRNA, the mRNA sequence coding for the signal peptide of apo A-I, and the sequence coding for the mature protein, respectively. In addition, the nucleotide sequence analysis of the apo A-I gene allowed determination of the complete amino acid sequence of the primary translation product of apo A-I mRNA. This amino acid sequence consists of 267 residues including a 24-residue-long amino-terminal extension (preprosegment). Finally, we show that the apo A-I gene contains six 66-base-pair-long tandemly repeated DNA segments, which suggests that the gene may have evolved by intragenic duplication events.

Apolipoprotein A-I↗

PubMed1983

Intracellular and extracellular processing of human apolipoprotein A-I: secreted apolipoprotein A-I isoprotein 2 is a propeptide.

We have recently proposed that the major secreted isoprotein form of human apolipoprotein A-I (designated apo A-I2) is modified extracellularly to become the predominant apo A-I form seen in plasma (designated apo A-I4). In the current report we demonstrate that the primary translation product of human apo A-I (designated apo A-I2p) has a 24-amino-acid NH2-terminal extension with a sequence of Met-Lys-Ala-Ala-Val-Leu-Thr-Leu-Ala-Val-Leu-Phe- Leu-Thr-Gly-Ser-Gln-Ala-Arg-His-Phe-Trp-Gln-Gln. The first 18 amino acids of this NH2-terminal extension are cleaved intracellularly by the signal peptidase, resulting in the formation of apo A-I2, which is the secreted form of apo A-I. Sequence analysis of apo A-I2 confirmed that it contains a hexapeptide extension at its NH2 terminus compared to apo A-I4. This observation demonstrates that apo A-I2 is a propeptide and that the apo A-I2 to apo A-I4 conversion involves the removal of the NH2-terminal hexapeptide of apo A-I2 by a protease in plasma, lymph, or both. Our findings indicate that apo A-I is synthesized as a prepropeptide, which undergoes intracellular and extracellular proteolysis to attain the major plasma apo A-I4 isoprotein form.

Amino Acid Sequence↗

PubMed1982

Identification and DNA sequence of a human apolipoprotein E cDNA clone.

cDNA clones encoding human apolipoprotein E were identified by screening an adult human liver cDNA library with an oligonucleotide probe. The probe was a mixture of synthetic 14-base long DNA oligomers constructed to correspond to all possible codons for apo-E amino acids 218-222. Plasmids from four of the 20 clones selected by this screening procedure were digested with PstI and all had five internal PstI sites with a total length of the cDNA insert of approximately 900 base pairs. DNA sequence analysis of one of these clones, designated pE-301, revealed that it corresponded to apo-E amino acids 81-299, and contained a standard termination codon, polyadenylation signal, and poly A tail. The DNA sequence examined included the known apo-E polymorphic sites at amino acids 112, 145, and 158, and the mutant apo-E phenotypes can all be explained on the basis of a single base substitution in the first position of each of these codons. This work supports the hypothesis that the apo-E polymorphism is due to mutations in the region of DNA coding for the apo-E structural gene.

Adult↗

PubMed1982

Isolation and characterization of cDNA clones for human apolipoprotein A-I.

We have isolated cDNA clones encoding human apolipoprotein (apo) A-I. Twenty putative apo A-I cDNA clones were selected by screening 10,000 clones of an adult human liver cDNA library with an oligonucleotide probe. The probe was a mixture of synthetic 14-base-long DNA oligomers constructed to correspond to the codons for apo A-I amino acids 105-109. Four of these clones were examined further and showed 600- to 800-base-pair (bp) inserts. Preliminary restriction mapping and partial DNA sequence analysis indicated that the shorter inserts were a subset of the longer DNA inserts. DNA sequence analysis of the clone with an insert of approximately equal to 600 bp, designated pAI-113, revealed that it contained a DNA sequence corresponding to apo A-I amino acids 94-243. The DNA base sequence of this clone also contained a standard termination codon, polyadenylylation signal, and poly(A) tail. Partial DNA sequence of a second clone that contained an 800-bp insert, designated pAI-107, showed that it corresponded to apo A-I amino acids 18-243 and also included the 3' untranslated region. Isolation of these cDNA clones will facilitate molecular analyses of apolipoproteins in normal and disease states.

Amino Acid Sequence↗

PubMed1981

Effects of (hydroxymethyl)trimethylpsoralen on structure and function of bacteriophage MS2 ribonucleic acid.

Treatment of bacteriophage MS2 with 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen and 360-nm light caused a dose-dependent decline in the infectivity of the virus. Covalent photobinding of a single psoralen molecule on the phage genome was a lethal event. Ribonucleic acid (RNA) extracted from psoralen and light-treated virus had a dose-dependent 385-nm fluorescence emission but was unaltered in its physical properties compared to control RNA samples. Phage adsorption and penetration in Escherichia coli host cells were unaffected, but in vivo replication of the treated virus was affected to the same extent as infectivity. The cell-free translational activity of the MS2 RNA was also severely reduced after psoralen and light treatment of the phage. Examination of the in vitro translation products revealed that the synthesis of the viral replicase protein was most substantially affected. Psoralen treatment of purified, protein-free MS2 RNA promoted an even greater reduction in cell-free synthesis of all viral proteins. This difference in translational function was consistent with the observation that virion-free RNA bound approximately 4 times as much psoralen as did RNA treated within the phage capsid. It was concluded that the replicase gene is the most sensitive region of the viral RNA molecule for psoralen binding.

Coliphages↗

PubMed1979

Properties of Escherichia coli 16S ribosomal ribonucleic acid treated with 4,5',8-trimethylpsoralen and light.

16S rRNA reacted with the furocoumarin 4,5',8-trimethylpsoralen (trioxsalen) and 360-nm light showed a number of chemical and physical differences from untreated RNA. After extensive irradiation, five molecules of trioxsalen were bound per molecule of RNA. The trioxsalen-treated RNA had an altered ultraviolet absorption spectrum and a distinctive fluorescence emission spectrum. The modified RNA was significantly more resistant to T1 ribonuclease digestion than was control RNA. Treated RNA, when mixed with purified ribosomal proteins, was not functional in the in vitro reconstitution of 30S subunits and yielded more slowly sedimenting particles which were inactive in protein synthesis assays. By contrast, 16S rRNA within the 30S subunit structure did not exhibit these changes when reacted with the same dose of trioxsalen and light, suggesting that the ribosomal proteins were effective in protecting the RNA from interaction with the drug.

Cell Fractionation↗

PubMedDate not supplied

Linkage of human apolipoproteins A-I and C-III genes.

It has recently been suggested that polymorphisms in the human apolipoprotein A-I (apo A-I) gene locus may be related to the development of premature atherosclerosis and hypertriglyceridaemia. To understand if and how these polymorphisms affect apo A-I gene expression, we studied the genomic sequences flanking the apo A-I gene. Here we show the presence of another apolipoprotein gene, apolipoprotein C-III (apo C-III), approximately 2.6 kilobases (kb) downstream of the 3' end of the apo A-I gene. We also show that the apo A-I and apo C-III genes are convergently transcribed and that a polymorphism previously reported to be associated with hypertriglyceridaemia may be due to a single base pair substitution in the 3'-noncoding region of apo C-III mRNA.

Amino Acid Sequence↗

PubMedDate not supplied

A DNA insertion in the apolipoprotein A-I gene of patients with premature atherosclerosis.

Apolipoprotein A-I (apo A-I) is the major protein constituent of high-density lipoprotein (HDL). The study of the apo A-I gene is of interest because plasma levels of HDL have been inversely correlated with the development of coronary artery disease and because polymorphisms related to this gene have been associated with hypertriglyceridaemia and premature atherosclerosis. We have recently isolated and characterized the human apo A-I gene and have shown that apo A-I and apolipoprotein C-III (apo C-III) genes are physically linked and that a polymorphism (of unknown frequency in the general population) of the apo A-I gene is inherited as a mendelian trait linked to premature atherosclerosis in an affected family (not the same polymorphism as has previously been reported to be associated with hypertriglyceridaemia). Here we report that this polymorphism is due an at least 6.5-kilobase (kb) DNA insertion in the coding region of the apo A-I gene and that there is no detectable alteration (as determined by limited genomic blotting analysis) of the apo C-III gene of these patients.

Apolipoprotein A-I↗

PubMedDate not supplied

Human apolipoprotein A-I--C-III gene complex is located on chromosome 11.

The genes for two of the apolipoproteins, apo A-I and apo C-III, previously shown to be within 3kb in the genome, were localized to human chromosome 11 by Southern blot analysis of DNA from human-rodent somatic cell hybrids. These two genes were shown to exhibit polymorphisms associated with dyslipoproteinemia and premature atherosclerosis, and it will now be possible to examine the relationship of these genes to the many others that have been assigned to this chromosome.

Animals↗