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Biomedical subjects

J I Gordon

Publications and source records attributed to J I Gordon.

At least 271 records · Page 15Linked to original sources
PubMed1984

Cloning of a cDNA encoding rat intestinal fatty acid binding protein.

Intestinal fatty acid binding protein mRNA is one of the most abundant mRNA species in the rat small intestinal epithelium. RNA transfer blot analyses disclosed that the mRNA encoding intestinal fatty acid binding protein is approximately equal to 900 nucleotides long and not represented in liver RNA. We have identified 564 nucleotides of this mRNA, including 12 nucleotides of the 5' nontranslated region, the coding portion, and 155 nucleotides of the 3' nontranslated domain. The primary translation product encoded by this mRNA contains 132 amino acids and has a Mr of 15,062. The derived protein sequence was verified by automated sequential Edman degradation of the intact polypeptide isolated from a wheat germ cell-free system. The in vitro product is NH2-terminally acetylated, a finding that is consistent with its ultimate cytoplasmic destination. Comparison of the amino acid sequence of this protein with liver fatty acid binding protein, a polypeptide specified by the most abundant small intestinal epithelial mRNA, revealed significant homology and similarity in the predicted secondary structures of their NH2-terminal domains.

Amino Acid Sequence↗
PubMed1984

Comparative in vitro study of the pro-apolipoprotein A-I to apolipoprotein A-I converting activity between normal and Tangier plasma.

We examined the ability of the plasma of a 52-yr-old male Tangier patient to effect the conversion of radiolabeled pro-apolipoprotein A-I (apo A-I), isolated from hepatoma cell culture media, into mature apo A-I. The conversion was assessed by amino-terminal sequence analysis, isoform patterns with two-dimensional gel electrophoresis, and a rapid assay based on the different solubilities of intact pro-apo A-I and its hexapeptide prosegment in 10% trichloroacetic acid. We found that the converting activity of Tangier plasma was comparable to that exhibited by control normolipidemic plasma and that in both cases pro-apo A-I was correctly processed at the Gln-Asp bond. After ultracentrifugal fractionation of Tangier plasma at d = 1.21 g/ml, the pro-apo A-I-to-mature apo A-I converting activity was mainly recovered in the middle fraction of d = 1.225 g/ml and was at least 10-fold more effective than the top and bottom fractions. In contrast, in normal plasma the activity was only present in the top and bottom fractions. It has been previously established that in Tangier plasma the pro-apo A-I/apo A-I ratio is significantly higher than normal (1 vs. 0.02). Our studies suggest that this abnormal ratio is not the result of a reduced converting enzyme activity and may relate to differences in turnover rates between Tangier and normal plasma apolipoproteins.

Amino Acid Sequence↗
PubMed1984

Processing of rat liver apoprotein E primary translation product.

The primary translation product of rat liver apoE mRNA was isolated from wheat germ cell-free translation systems. Plasma apoE and the primary translation product migrated similarly on SDS-polyacrylamide gels, had similar partial proteolytic peptide maps, and bound to and coeluted from heparin-Sepharose columns. Comparison of the partial amino acid sequence of the primary translation product with the amino-terminal sequence of plasma apoE indicated that rat apoE is initially synthesized with an 18 amino acid amino-terminal extension. This entire segment was removed cotranslationally by canine microsomes possessing signal peptidase activity. The microsome-processed translation product did not contain an endoglycosidase H-sensitive oligosaccharide, suggesting that rat apoE is O-glycosylated.

Amino Acid Sequence↗
PubMed1984

Apolipoproteins of human plasma high density lipoproteins. Biology, biochemistry and clinical significance.

The steps involved in assembly, secretion and intravascular remodeling of plasma high density lipoproteins (HDL) are still largely unknown. However, recent progress has been made in understanding the biosynthesis of the major HDL-apolipoproteins, apo A-I and apo A-II. Combined with information about apoprotein gene structure, it has been possible to identify molecular lesion(s) associated with some clinical disorders of lipoprotein metabolism.

Apolipoprotein A-I↗
PubMed1983

Biosynthesis of human preproapolipoprotein A-II.

The primary translation product of human apolipoprotein A-II was purified from wheat germ and ascites cell-free lysates programmed with RNA isolated from either a hepatocellular carcinoma cell line (HepG2) or intestinal epithelium. A-II mRNA represents 0.2% of the translatable RNA in these hepatocytes and in jejunal epithelium. Plasma high density lipoprotein-associated A-II is a 77-amino acid polypeptide. The primary translation product is 100 amino acids long and contains a 23-amino acid NH2-terminal extension. Cotranslational cleavage of the cell-free product indicated that this NH2-terminal sequence consists of an 18-amino acid long signal peptide, Met-Lys-Leu-Leu-Ala-Ala-X-Val-Leu-Leu-Leu-X-X-Cys-X-Leu-X-X-, and a 5-amino acid long propeptide, Ala-Leu-Val-Arg-Arg. This functional division was confirmed by sequencing the stable intracellular form of apolipoprotein A-II isolated from HepG2 cells. Approximately 45% of the proapo-A-II is cleaved to the mature form during export from HepG2 cells. The COOH-terminal dipeptide conforms to the rule that prosegments are cleaved after paired basic residues. We have previously shown (Gordon, J. I., Sims, H. F., Lentz, S. R., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1983) J. Biol. Chem. 258, 4037-4044) that proapolipoprotein A-I is not cleaved during export from these cells and contains a prosegment with a COOH-terminal Gln-Gln dipeptide. Therefore, proteolytic processing of the two principal high density lipoprotein-associated apolipoproteins proceeds along different pathways.

Amino Acid Sequence↗
PubMed1983

In vitro conversion of proapoprotein A-I to apoprotein A-I. Partial characterization of an extracellular enzyme activity.

Previous studies have established that human hepatocellular carcinoma cells (Hep G2) secrete into serum-free medium the pro form of apolipoprotein A-I (proapo-A-I) suggesting that its conversion to mature apo-A-I occurs after secretion. In order to assess the mode and site of proapo-A-I to apo-A-I conversion, we incubated the medium from [3H]proline-labeled Hep G2 cells with either human plasma, serum, lymph, or fractions thereof obtained by density gradient ultracentrifugation. The conversion was monitored by two-dimensional gel electrophoresis and by Edman degradation. Human plasma, serum, or mesenteric lymph all induced proapo-A-I to apo-A-I conversion; this was time dependent, unaffected by the serine protease inhibitor phenylmethylsulfonyl fluoride and inhibited by EDTA. Purified radiolabeled proapo-A-I bound to lymph chylomicrons and plasma high density lipoproteins. The converting enzyme was associated with both of these particles. Activity was also found in the d greater than 1.21-g/ml fraction and may have been derived from high density lipoprotein after displacement by high salts and/or ultracentrifugal force. We conclude that the conversion of proapo-A-I to apo-A-I occurs extracellularly and is probably effected by a metallo-enzyme which may act at the amphiphilic surface of either chylomicrons or high density lipoproteins.

Amino Acid Sequence↗
PubMed1983

Proteolytic processing of human preproapolipoprotein A-I. A proposed defect in the conversion of pro A-I to A-I in Tangier's disease.

The primary translation product of human intestinal apolipoprotein A-I mRNA was isolated from wheat germ and ascites cell-free translation systems. Comparison of its NH2-terminal sequence with that of plasma high density lipoprotein-associated A-I showed that it is initially synthesized as a preproprotein. Like rat preproapolipoprotein A-I, it contains an 18-amino acid prepeptide and a 6-amino acid propeptide. The highly unusual COOH-terminal Gln-Gln dipeptide present in the rat pro-segment is also represented at the same position in the human sequence. The functional division of the 24-amino acid NH2-terminal extention into pro- and presegments was verified by finding that the stable intracellular form of A-I in a human hepatoma cell line was the proprotein. Edman degradation of radiolabeled intracellular and extracellular A-I indicated that this apolipoprotein was secreted without proteolytic cleavage of its hexapeptide prosegment. Therefore, it appears that apolipoprotein A-I undergoes an additional proteolytic processing step before it is fully integrated into plasma high density lipoprotein. Two-dimensional gel electrophoresis of purified proapolipoprotein A-I isolated from the hepatocyte cell culture media indicated that it corresponds to isoforms 2 and 3, the basic A-I isoproteins which are the precursors of plasma A-I and the predominant plasma A-I isoforms found in patients with Tangier's disease (Zannis, V. I., Lees, A. M., Lees, R. S., and Breslow, J. L. (1982) J. Biol. Chem., 257, 4978-4986). Therefore this pathologic state probably arises from a defect in the conversion of proapolipoprotein A-I to apolipoprotein A-I.

Amino Acid Sequence↗
PubMed1983

The nucleotide sequence of rat liver fatty acid binding protein mRNA.

Rat liver fatty acid binding protein mRNA is present in both liver and intestinal epithelium. It is the most abundant mRNA in the small intestinal mucosa. We have determined the sequence of this mRNA from cloned cDNA. It consists of a 5'-nontranslated domain at least 39 nucleotides long, a coding region which specifies a 127-amino acid, 14,184-dalton polypeptide, and a 3'-nontranslated region 68 nucleotides long. An AAUAAA sequence begins 21 nucleotides before the 3'-terminal poly(A) tail. The derived protein sequence was verified by automated sequential Edman degradation of (i) the primary translation product of the mRNA and (ii) a cyanogen bromide peptide generated from the mature liver protein. Evidence is presented which suggests that this protein is targeted to the cytoplasm. The NH2 terminus of the primary translation product is acetylated in vitro. No cleavable signal peptide sequence or internal signal sequence equivalent was detectable in a co-translational cleavage assay. Cloned cDNA was used to establish that this mRNA was 12 times more abundant in intestinal epithelial than in liver RNA.

Amino Acid Sequence↗
PubMed1983

Regulation of creatine phosphokinase expression during differentiation of BC3H1 cells.

The intracellular mechanisms involved in the regulation of creatine phosphokinase expression in the BC3H1 muscle-like cell line have been examined under conditions of enzyme induction and repression. In the presence of low serum concentrations, BC3H1 cells cease to grow and synthesize high levels of creatine phosphokinase. When differentiated BC3H1 cultures are exposed to media containing high serum concentrations, cell division is reinitiated and further induction of creatine phosphokinase is inhibited. Accumulation of creatine phosphokinase-mRNA appears to be intimately coupled to the state of growth of BC3H1 cells. Log phase cells do not contain detectable levels of translatable creatine phosphokinase-mRNA; however, following cessation of growth, creatine phosphokinase-mRNA accumulates in approximate proportion to the increase in creatine phosphokinase activity. Reinitiation of cell division in quiescent differentiated cultures results in the arrest of further accumulation of creatine phosphokinase-mRNA but does not inhibit the translation of pre-existing creatine phosphokinase-mRNA. Under conditions of enzyme repression, however, the newly synthesized creatine phosphokinase appears to be enzymatically inactive. These results indicate that the expression of the muscle phenotype in BC3H1 cells is regulated by components present in serum and that myogenic differentiation is at least partially reversible following re-entry of quiescent cells into the cell cycle.

Animals↗
PubMed1982

Cloning of a complementary deoxyribonucleic acid encoding a portion of rat intestinal preapolipoprotein AIV messenger ribonucleic acid.

Apolipoprotein AIV is one of the principal apolipoproteins synthesized by the rat small intestine. We have cloned a cDNA encoding a portion of preapolipoprotein AIV mRNA. A kinetically fractionated cDNA probe highly enriched for the abundant intestinal mucosal mRNA sequences was used to screen a library of recombinants containing cDNA generated from total intestinal epithelial mRNA. The abundant class of mRNA sequences was defined by hybridization analyses. This frequency class had an aggregate complexity of 5300 nucleotides and represented 25% of accumulated mRNA sequences.l The mRNAs comprising this class were identified by in vitro translation and included preapolipoprotein AIV, preproapolipoprotein AI, intestinal fatty acid binding protein, and liver fatty acid binding protein. A cDNA-containing clone derived from preapolipoprotein AIV mRNA was identified among probe-positive recombinants. This cDNA was used to establish that apolipoprotein AIV mRNA has a mass of 550000 daltons (equivalent to 1780 nucleotides) and represents 0.013% of total cellular RNA in the fasting state. Acute feeding with triglyceride-rich meals resulted in a 2-fold increase in preapolipoprotein AiV mRNA after 4 h.

Animals↗
PubMed1982

Proteolytic processing of the primary translation product of rat intestinal apolipoprotein A-IV mRNA. Comparison with preproapolipoprotein A-I processing.

Total cellular RNA, isolated from rat intestinal epithelium, was translated in a wheat germ cell-free system, and the primary translation product of apolipoprotein A-IV mRNA was purified by immunoprecipitation with monospecific antibody. NH2-terminal sequence analyses of mature rat plasma high density lipoprotein-associated A-IV and the cell-free product revealed that A-IV is initially synthesized with a 20-amino-acid NH2-terminal extension. Co-translational cleavage of the primary translation product indicated that the entire NH2-terminal extension behaved as a prepeptide. The prepeptides of intestinal apolipoproteins A-I and A-IV have 59% sequence homology. However, A-IV does not possess the unusual propeptide previously identified in intestinal A-I (Gordon, J. I., Smith, D. P. Andy, R., Alpers, D. H. Schonfeld, G., and Strauss, A. (1982) J. Biol. Chem. 257, 971-978). Therefore it appears that proteolytic processing of these two apolipoproteins differs significantly.

Amino Acid Sequence↗
PubMed1982

The primary translation product of rat intestinal apolipoprotein A-I mRNA is an unusual preproprotein.

We have characterized the NH2-terminal sequence of the primary translation product of intestinal apolipoprotein A-I mRNA. Co-translational cleavage of this in vitro product and NH2-terminal sequence analysis of plasma high density lipoprotein-associated apolipoprotein A-I showed that it is initially synthesized as a preproprotein. The 24-amino-acid NH2-terminal extension consists of an 18-amino-acid presegment; Met-Lys-Ala-Ala-Val-Leu-Ala-Val-Ala-Leu-Val-Phe-Leu-Thr-Gly-Cys-Gln-Ala, and a hexapeptide prosegment; -X-Glu-Phe-X-Gln-Gln, followed by the NH2 terminus of the "mature" plasma protein: Asp-Glu-Pro-Gln-X-Gln-Trp-Asp-Arg-Val-Lys-Asp-Phe-Ala-X-Val-Tyr-X-Asp-Ala-Val. Although the prepeptide resembles other signal peptides, the prosegment differs from other propeptides in that it does not terminate with paired basic amino acid residues. These results suggest that translocation of nascent preproapolipoprotein A-I is similar to that of other secretory proteins. However, post-translational proteolytic processing of proapolipoprotein A-I must be unique, and could play a role in the formation of nascent high density lipoprotein particles.

Amino Acid Sequence↗
PubMed1980

Coordinate regulation of two estrogen-dependent genes in avian liver.

Livers of egg-laying species contain abundant mRNAs encoded by both estrogen-responsive and constitutively expressed genes. We have recently constructed cDNA clones from three members of the abundant mRNA class of hen liver. One of these mRNA species was identified as serum albumin mRNA, and another as vitellogenin mRNA. In this study we have identified the third member of the group as apo VLDLII mRNA. Hybridization analyses using cloned cDNA probes indicate that expression of the apo VLDLII gene in rooster liver, like that of the vitellogenin gene, is completely dependent upon the administration of estrogen. The apo VLDLII and vitellogenin genes appear to be the only genes capable of high rates of expression in the liver that exhibit such an exceptional response to the hormone. Administration of estrogen resulted in the appearance of both mRNA species within 30 min, followed by a rapid accumulation to several thousand copies per cell. Removal of the hormone caused a marked destabilization of both vitellogenin mRNA and apo VLDLII mRNA. In contrast, the absolute levels of serum albumin mRNA were unaffected by the hormone. Comparative studies on the structure and organization of these three genes may reveal elements involved in determining their rates of expression in the presence and absence of estrogen.

Animals↗
PubMed1979

Specific transcription in chicken liver chromatin by endogenous RNA polymerase II. Comparison of an estrogen-inducible gene with a constitutively expressed gene.

We have developed a system for the in vitro transcription of specific genes in rooster liver chromatin by endogenous RNA polymerase II that maintains the specificity of transcription in vivo. Radioactive transcripts synthesized in vitro were identified and quantitated by hybridization to a vast excess of cloned cDNA. The cDNA preparations employed corresponded to vitellogenin mRNA, the synthesis of which is responsive to estrogen stimulation in vivo, and chicken serum albumin mRNA, the synthesis of which is not significantly affected by estrogen stimulation in vivo. Comparing the pattern of transcription of the albumin and vitellogenin genes in chromatin from the liver of the normal rooster with the pattern in chromatin from the liver of the estrogen-stimulated rooster, we found that prior estrogen treatment of the rooster is attended by a slight decrease in the differential rate of transcription of the albumin gene and approximately a 10-fold increase in the differential rate of transcription of the vitellogenin gene. Because this pattern of transcription reflects the estrogen-induced changes in transcription observed in vivo, chromatin preparations from the livers of normal and estrogen-stimulated roosters can be used to investigate regulation of specific gene transcription at the molecular level in vitro.

Amanitins↗
PubMed1978

Cloning of a double-stranded cDNA that codes for a portion of chicken preproalbumin. A general method for isolating a specific DNA sequence from partially purified mRNA.

A scheme is presented for cloning a double-stranded cDNA molecule that codes for a portion of chicken preproalbumin. This method, which does not require pure mRNA or cDNA, has widespread applicability. Chicken preproalbumin was identified as a Mr = 72,000 polypeptide by immunoprecipitation of proteins synehesized in a wheat germ cell-free translation system from total, guanidine.HCl-extracted, rooster liver RNA. After removal of the bulk of the ribosomal RNA by poly(U)-Sephadex G-10 chromatography, albumin mRNA was enriched approximately 2-fold by centrifugation through low salt, isokinetic sucrose gradients, until it represented about 30% of the mRNA sequences present. Double-stranded cDNA prepared from this mRNA was then inserted into the Pst 1 site of the plasmid PBR322 by the "G-C tailing" technique and the recombinant DNA was used to transform Echerichia coli stran X1776. Transformants containing putative albumin DNA sequences were identified by colony hybridization with a cDNA probe that was highly enriched for albumin cDNA sequences. This probe was isolated by hybridizing the partially purified RNA preparation to its cDNA, under conditions of RNA excess, to a R0t value such that only the most abundant cDNA sequences had hybridized. Unhybridized, less abundant, sequences were destroyed by subsequent S1 nuclease digestion. The identity of clones that hybridized to this abundant class cDNA was established by DNA-mRNA hybrid-arrested cell-free translation. Hybridization of nick-translated, albumin-containing, plasmid DNA to total liver poly(A)+ RNA, that had been separated on methyl mercury agarose gels and transferred to diazobenzyloxymethyl paper, established that avian albumin mRNA has a molecular weight of 850,000. This molecular weight corresponds to approximately 2,600 nucleotides, or 600 nucleotides longer than the size required to code for the preproalbumin polypeptide.

Animals↗
PubMed1978

Primary induction of vitellogenin mRNA in the rooster by 17beta-estradiol.

We have studied the kinetics of vitellogenin mRNA accumulation in rooster liver after a primary injection of 17beta-estradiol. The levels of vitellogenin mRNA have been determined both by hybridization of total cellular RNA to vitellogenin cDNA and by translation of vitellogenin mRNA in a wheat germ cell-free system. The results obtained by both methods of analysis are in good agreement and indicate that vitellogenin mRNA is present in the liver of normal roosters at a level of 0-5 molecules per liver cell and increases in amount during the 3 days following injection of estrogen, reaching a level of almost 6000 molecules per cell at the peak of the response. The level of vitellogenin mRNA declined exponentially during the next 14 days with a half-life of 29 hr, reaching a level of less than 10 molecules per cell at 17 days after injection of the hormone. The levels of vitellogenin mRNA after stimulation with estrogen have been correlated with the in vivo rate of synthesis of the vitellogenin polypeptide. The results indicate that the rate of vitellogenin synthesis is closely correlated with the level of vitellogenin mRNA. On the basis of these findings, we conclude that vitellogenin mRNA does not exist in the liver in an untranslated form after withdrawal from estrogen.

Animals↗
PubMed1977

Kinetics of avian vitellogenin messenger RNA induction. Comparison between primary and secondary response to estrogen.

Following either primary or secondary stimulation of cockerels with 17beta-estradiol, vitellogenin mRNA begins to accumulate in the liver after about 30 min, reaches a maximum in 3 days, and decays thereafter with a half-life of 30 h. During primary induction, accumulation of vitellogenin mRNA begins at a low rate (50 nucleotides/s/nuclear equivalent of DNA) and after 4 h, shifts to a higher rate (340 nucleotides/s/nuclear equivalent of DNA). In contrast, during secondary induction (administration of estrogen several weeks after the primary response has ceased), accumulation of vitellogenin mRNA begins at the rate of 350 nucleotides/s/nuclear equivalent of DNA and subsequently increases by about 40%. These accumulation rates result in a maximal level of vitellogenin mRNA that is approximately 1.5 times higher during secondary stimulation than that found during primary stimulation. This difference is sufficient to explain the anamnestic response to secondary hormonal stimulation that results in higher levels of circulating vitellogenin in the plasma of the rooster.

Animals↗