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Biomedical subjects

D W Chung

Publications and source records attributed to D W Chung.

At least 55 records · Page 3Linked to original sources
PubMed1989

Circulating human blood platelets retain appreciable amounts of poly (A)+ RNA.

Platelets lack a nucleus and are usually considered to be incapable of protein synthesis due to an apparent lack of messenger RNA, precluding the construction of platelet cDNA libraries and hindering the cloning of authentic platelet cDNA's. We reasoned that vestigial amounts of messenger RNA may remain in platelets when they first separate from the megakaryocyte and circulate in the peripheral blood. We isolated poly (A)+ RNA from platelets obtained by pheresis of individuals with elevated blood platelet counts due to a myeloproliferative syndrome termed essential thrombocythemia. Northern blots using probes for platelet glycoprotein Ib indicate that the poly (A)+ RNA obtained from the platelets of these donors is, in fact, derived from platelets. Cell free translation studies using the platelet poly (A)+ RNA indicate that the material is translationally active. We conclude that, contrary to prevailing information, circulating human blood platelets retain appreciable amounts of poly (A)+ RNA and that this RNA can be harvested by the described approach. The poly (A)+ RNA provides templates for the synthesis of cDNA's that code for platelet proteins.

Blood Platelets↗
PubMed1989

The molecular genetics of factor XI deficiency.

Hereditary factor XI deficiency is characterised by a functional deficiency of factor XI and the absence of factor XI-related antigen in circulation. It occurs with a high frequency in the Ashkenazi Jewish population. Cloning of abnormal factor XI genes and studies on the molecular genetics of factor XI deficiency show that the cause for factor XI deficiency is heterogeneous. So far, two independent single base substitutions, one at the conserved intron donor consensus dinucleotide of intron N (type I) and a nonsense mutation at the codon for Glu117 (type II), have been identified. These two types of mutation together account for approximately half of the genetic changes in abnormal factor XI genes. At least one or more types of genetic change has yet to be defined. In the course of these studies, rapid methods that utilize the polymerase chain reaction and subsequent restriction endonuclease analysis have been developed.

Amino Acid Sequence↗
PubMed1989

Factor XI (plasma thromboplastin antecedent) deficiency in Ashkenazi Jews is a bleeding disorder that can result from three types of point mutations.

Factor XI (plasma thromboplastin antecedent) deficiency is a blood coagulation abnormality occurring in high frequency in Ashkenazi Jews. Three independent point mutations that result in a blood coagulation abnormality have been identified in the factor XI gene of six unrelated Ashkenazi patients. These mutations either disrupt normal mRNA splicing (type I), cause premature polypeptide termination (type II), or result in a specific amino acid substitution (type III). The three different genotypes were present in the six patients as type I/II, type II/III, and type III/III. Thus far no correlation was found between the three genotypes and the bleeding tendency in these patients.

Adult↗
PubMed1988

The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence.

The primary structure of the beta chain of human glycoprotein Ib (GPIb), the platelet receptor for von Willebrand factor, has been established by a combination of cDNA cloning and amino acid sequence analysis. A lambda phage cDNA expression library prepared from human erythroleukemia cells (HEL cells) was screened with a radiolabeled affinity-purified rabbit polyclonal antibody to the beta chain of GPIb. Eighteen positive clones were isolated and plaque-purified and the nucleotide sequences of three were determined. The composite sequence spanned 968 nucleotides and included a 5' untranslated region of 22 nucleotides, an open reading frame of 618 nucleotides encoding a signal peptide of 28 amino acids and a mature protein of 181 amino acids, a stop codon, and a 3' noncoding region of 307 nucleotides. The 3' noncoding sequence also contained a polyadenylylation signal (AATAAA) 14 nucleotides upstream from the poly(A) tail of 18 nucleotides. Edman degradation of the intact beta chain and of peptides produced by chemical cleavage yielded amino acid sequences spanning 76 residues that were identical to those predicted from the cDNA. The amino-terminal region of the beta chain contains a leucine-rich sequence of 24 amino acids that is similar to a sequence that occurs as seven tandem repeats in the alpha chain of GPIb and nine tandem repeats in leucine-rich alpha 2-glycoprotein. The leucine-rich sequence in the beta chain of GPIb is flanked on both sides by amino acid sequences that are similar to those flanking the leucine-rich tandem repeats of the alpha chain of GPIb and leucine-rich alpha 2-glycoprotein. The amino-terminal region of the beta chain of GPIb is followed by a transmembrane segment of 25 amino acids and an intracellular segment of 34 amino acids at the carboxyl terminus of the protein. The intracellular segment contains an unpaired cysteine and two potential sites for phosphorylation by cAMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1987

Organization of the gene for human factor XI.

Factor XI (plasma thromboplastin antecedent) is a plasma glycoprotein that participates in the early phase of blood coagulation. The gene for the human protein has been isolated from two different lambda phage genomic libraries. Four independent recombinant lambda phage carrying overlapping DNA inserts that coded for the entire gene for factor XI were isolated and characterized by restriction mapping, Southern blotting, and selective DNA sequencing to establish the number and location of the intron-exon boundaries. The gene for human factor XI was 23 kilobases in length and consisted of 15 exons (I-XV) and 14 introns (A-N). Exon I coded for the 5' untranslated region, and exon II coded for the signal peptide. The next eight exons (III-X) coded for the four tandem repeats of 90 or 91 amino acids that were present in the amino-terminal region of the mature protein. Each of these tandem repeats was coded by two exons that were interrupted by a single intron, and these introns were located in essentially the same position within each of the four tandem repeats. The carboxyl-terminal region of the protein, which contained the catalytic chain, was coded by five exons (XI-XV) that were interrupted by four introns. The last four introns were located in the same positions as those in the genes for human tissue plasminogen activator and human urokinase.

Amino Acid Sequence↗
PubMed1987

Cloning of the alpha chain of human platelet glycoprotein Ib: a transmembrane protein with homology to leucine-rich alpha 2-glycoprotein.

Glycoprotein Ib is a surface membrane glycoprotein of platelets that functions as a receptor for von Willebrand factor. It is a heterodimer composed of an alpha and a beta chain linked by a disulfide bond(s). A phage lambda gt11 cDNA expression library prepared from mRNA from a human erythroleukemia cell line, HEL, was screened using an affinity-purified antibody to the glycocalicin portion of the alpha chain of glycoprotein Ib. Eleven positive clones were isolated and plaque-purified. The largest cDNA insert was 2420 nucleotides in length and coded for a leader sequence of 16 amino acids, a mature protein of 610 amino acids, and a stop codon. It also contained 42 nucleotides of 5' noncoding sequence and 497 nucleotides of 3' noncoding sequence, including a poly(A) tail. The amino acid sequence of the alpha chain of GPIb predicted from the cDNA agreed completely with the sequence of 156 amino acids that was determined by Edman degradation of peptides isolated from human platelet glycocalicin after digestion with trypsin or Staphylococcus aureus V8 protease. The extracytoplasmic domain of the alpha subunit of GPIb contains several noteworthy structural features, including a region of seven tandem repeats of 24 amino acids that are homologous with those present in leucine-rich alpha 2-glycoprotein. The extracytoplasmic domain also contains two hydrophilic regions, one rich in charged amino acids and a second rich in serine and threonine residues. The region rich in serine and threonine includes five repeats of nine amino acids as well as the majority of the O-linked carbohydrate sites present in the molecule. The extracytoplasmic domain is followed by a potential transmembrane segment of approximately 29 amino acids and a potential intracellular domain of approximately 100 amino acids located at the carboxyl end of the molecule.

Amino Acid Sequence↗
PubMed1986

Human plasma prekallikrein, a zymogen to a serine protease that contains four tandem repeats.

The amino acid sequence of human plasma prekallikrein was determined by a combination of automated Edman degradation and cDNA sequencing techniques. Human plasma prekallikrein was fragmented with cyanogen bromide, and 13 homogeneous peptides were isolated and sequenced. Cyanogen bromide peptides containing carbohydrate were further digested with trypsin, and the peptides containing carbohydrate were isolated and sequenced. Five asparagine-linked carbohydrate attachment sites were identified. The sequence determined by Edman degradation was aligned with the amino acid sequence predicted from cDNAs isolated from a lambda gt11 expression library. This library contained cDNA inserts prepared from human liver poly(A) RNA. Analysis of the cDNA indicated that human plasma prekallikrein is synthesized as a precursor with a signal peptide of 19 amino acids. The mature form of the protein that circulates in blood is a single-chain polypeptide of 619 amino acids. Plasma prekallikrein is converted to plasma kallikrein by factor XIIa by the cleavage of an internal Arg-Ile bond. Plasma kallikrein is composed of a heavy chain (371 amino acids) and a light chain (248 amino acids), and these 2 chains are held together by a disulfide bond. The heavy chain of plasma kallikrein originates from the amino-terminal end of the zymogen and is composed of 4 tandem repeats that are 90 or 91 amino acid residues in length. These repeat sequences are also homologous to those in human factor XI. The light chain of plasma kallikrein contains the catalytic portion of the enzyme and is homologous to the trypsin family of serine proteases.

Amino Acid Sequence↗
PubMed1986

Amino acid sequence of human factor XI, a blood coagulation factor with four tandem repeats that are highly homologous with plasma prekallikrein.

A lambda gtll cDNA library prepared from human liver poly(A) RNA has been screened with affinity-purified antibody to human factor XI, a blood coagulation factor composed of two identical polypeptide chains linked by a disulfide bond(s). A cDNA insert coding for factor XI was isolated and shown to contain 2097 nucleotides, including 54 nucleotides coding for a leader peptide of 18 amino acids and 1821 nucleotides coding for 607 amino acids that are present in each of the 2 chains of the mature protein. The cDNA for factor XI also contained a stop codon (TGA), a potential polyadenylation or processing sequence (AACAAA), and a poly(A) tail at the 3' end. Five potential N-glycosylation sites were found in each of the two chains of factor XI. The cleavage site for the activation of factor XI by factor XIIa was identified as an internal peptide bond between Arg-369 and Ile-370 in each polypeptide chain. This was based upon the amino acid sequence predicted by the cDNA and the amino acid sequence previously reported for the amino-terminal portion of the light chain of factor XI. Each heavy chain of factor XIa (369 amino acids) was found to contain 4 tandem repeats of 90 (or 91) amino acids plus a short connecting peptide. Each repeat probably forms a separate domain containing three internal disulfide bonds. The light chains of factor XIa (each 238 amino acids) contain the catalytic portion of the enzyme with sequences that are typical of the trypsin family of serine proteases. The amino acid sequence of factor XI shows 58% identity with human plasma prekallikrein.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence of the gene for the gamma chain of human fibrinogen.

A human genomic DNA library was screened for the gene coding for the gamma chain of fibrinogen by using a human cDNA for the gamma chain as a hybridization probe. The gene was identified in three overlapping recombinant lambda bacteriophage, and its sequence, including the immediate 5' and 3' flanking regions, was determined. The DNA sequence analysis revealed the presence of 10 exons coding for 411 amino acids present in the mature protein and a signal sequence of 26 amino acids. Two 30 base pair (bp) direct repeats of 93% identity were found 468 bp upstream from the transcription initiation site. The DNA sequence of the gene for the gamma chain of human fibrinogen showed considerable sequence homology with a partial sequence reported for the gene for the gamma chain of rat fibrinogen.

Amino Acid Sequence↗
PubMed1984

gamma and gamma' chains of human fibrinogen are produced by alternative mRNA processing.

cDNAs and the genomic DNA coding for the gamma and gamma' chains of human fibrinogen have been isolated and characterized by sequence analysis. The cDNAs coding for the gamma and gamma' chains share a common nucleotide sequence coding for the first 407 amino acid residues in each polypeptide chain. The predominant gamma chain contains an additional four amino acids on its carboxyl-terminal end (residues 408-411). These four amino acids, together with the 3' noncoding sequences, are encoded by the tenth exon. Removal of the ninth intervening sequence following the processing and polyadenylation reactions yields a mature mRNA coding for the predominant gamma chain. The less prevalent gamma' chain contains 20 amino acids at its carboxyl-terminal end (residues 408-417). These 20 amino acids are encoded by the immediate 5' end of the ninth intervening sequence. This results from an occasional processing and polyadenylation reaction that occurs within the region normally constituting the ninth intervening sequence. Accordingly, the gene for the gamma chain of human fibrinogen gives rise to two mRNAs that differ in sequence on their 3' ends. These mRNAs code for polypeptide chains with different carboxyl-terminal sequences. Both of these polypeptides are incorporated into the fibrinogen molecule present in plasma.

Amino Acid Sequence↗
PubMed1984

Characterization of a cDNA coding for human factor X.

A lambda gt11 cDNA library containing DNA inserts prepared from human liver mRNA has been screened with an antibody to human factor X, a plasma protein participating in the middle phase of the blood coagulation cascade. Ten positive clones were isolated from 2 X 10(6) phage and plaque purified. The cDNA in the phage containing the largest insert has been sequenced and shown to code for human factor X. This cDNA insert contained 1137 base pairs coding for a portion of the light chain of the molecule, a connecting region, the heavy chain, a stop codon, a short 3' noncoding region, and a poly(A) tail. The sequence of A-T-T-A-A-A, which functions as a potential recognition site for polyadenylylation or processing, was present in the 3' end of the coding sequence and preceded the stop codon of TGA by 1 base pair and the poly(A) tail by 14 base pairs. The amino acid sequence deduced from the cDNA indicated that factor X is synthesized as a single-chain polypeptide containing the light and heavy chains connected by an Arg-Lys-Arg tripeptide. The single-chain molecule is then converted to the light and heavy chains by cleavage of two (or more) internal peptide bonds. In plasma, these two chains are linked together by a disulfide bond. The DNA sequence coding for the active site of human factor X showed a high degree of identity with prothrombin and factor IX, two other vitamin K-dependent serine proteases that participate in blood coagulation. These data along with the protein sequence data previously published for the light chain of human factor X establish the complete amino acid sequence for the mature protein present in plasma.

Amino Acid Sequence↗
PubMed1983

Cloning of fibrinogen genes and their cDNA.

Cross-species hybridizations have enabled us to isolate and clone the gene for the beta chain of human fibrinogen. Highlights of the gene for the beta chain revealed by nucleotide sequence analyses, particularly in areas that have a direct bearing on defining the overall organization of the gene, have been presented. Nucleotide sequence determination has confirmed the presence of seven intervening sequences. The positions where several of these intervening sequences interrupt the coding region appear to be related to the functional domains of the polypeptide. A putative signal peptide has been identified. Studies on the cDNA for the human alpha chain indicate that the alpha chain polypeptide may be synthesized in a precursor form with a COOH-terminal extension of 15 amino acids as compared to the alpha chain present in the mature molecule found in plasma. We are in the process of isolating the genes for the alpha and gamma chains by a similar approach. We are hopeful that these studies will provide information as to how they are regulated and how they have undergone changes in the course of evolution.

Amino Acid Sequence↗
PubMed1983

Characterization of a complementary deoxyribonucleic acid coding for the alpha chain of human fibrinogen.

A human liver cDNA library was screened for the alpha chain of fibrinogen with a cDNA clone from the corresponding bovine molecule as a hybridization probe. Several human clones coding for the alpha chain were identified, and one of these was used to rescreen the entire cDNA library of 18 000 recombinants. Plasmids with the largest cDNAs were isolated, and their inserts were sequenced. The largest cDNA insert contained 2224 base pairs, including a noncoding region at the 5'-end followed by a region coding for a signal peptide of 19 (or 16) amino acids and a mature protein of 625 amino acids, a stop codon of TAG, another noncoding region, and a poly(A) tail at the 3'-end. Eight tandem repeats of 39 base pairs were observed starting with nucleotide 905 (amino acid residue 270) and ending with nucleotide 1213 (amino acid residue 372). The identity in the nucleotide sequence in the tandem repeats ranged from 72 to 95% when compared to a consensus sequence. The predicted amino acid sequence for the mature polypeptide chain was 15 amino acids longer at the carboxyl-terminal end than that of the alpha chain isolated and sequenced from plasma fibrinogen. This indicates that minor proteolysis has taken place on the carboxyl-terminal end of the alpha chains, and this modification has probably occurred during secretion or circulation of the protein in plasma.

Amino Acid Sequence↗
PubMed1983

Characterization of complementary deoxyribonucleic acid and genomic deoxyribonucleic acid for the beta chain of human fibrinogen.

A total of 148 cDNAs coding for the beta chain of human fibrinogen have been identified from a human liver cDNA library employing a bovine cDNA as a probe. The largest cDNA insert contained 1932 base pairs cloned into the PstI site of plasmid pBR322. This cDNA insert contained 66 base pairs coding for a portion or all of a signal sequence, 1383 base pairs coding for 461 amino acids in the mature protein, a stop codon of TAG, a noncoding region of 431 base pairs, and a poly(A) tail of 19 base pairs. Most of the cDNA inserts coding for the beta chain were found to have a noncoding region of 98 or 167 base pairs rather than 431 base pairs at the 3'-end. The bovine cDNA for the beta chain was also employed as a probe for screening a lambda phage library containing human genomic DNA. Seven positive phage were identified. One of the phage, which contained the entire gene for the beta chain of fibrinogen, was examined by electron microscopy, and portions of its DNA sequence are presented. Seven intervening sequences were identified in the gene for the beta chain of human fibrinogen. The largest intervening sequence (approximately 1.3 kilobases) was found at the 5'-end of the gene and was located between amino acid residues 8 and 9, which are present in fibrinopeptide B. A sequence analysis of the 5'-end of the gene also indicated that the B chain of human fibrinogen contained a signal sequence of either 16, 27, or 30 amino acid residues.

Amino Acid Sequence↗
PubMed1983

Characterization of a complementary deoxyribonucleic acid coding for the gamma chain of human fibrinogen.

A number of cDNAs coding for the gamma chain of human fibrinogen have been isolated from a liver cDNA library by employing a synthetic nucleotide mixture as a probe. One of the positive clones was then employed to screen the entire cDNA library of 18000 recombinants, yielding 320 positive clones for the gamma chain. The largest cDNA was 1638 base pairs in length and contained 10 base pairs of poly(G) at the 5'-end followed by 71 base pairs of noncoding nucleotides. The next 78 base pairs coded for a leader sequence that was 26 amino acids in length and included a methionine start signal and a typical hydrophobic core. The following 1233 base pairs coded for 411 amino acids that are present in the mature protein followed by a stop codon of TAA, 207 base pairs of noncoding nucleotides, a poly(A) track of 15 base pairs, and 22 base pairs of poly(C). Specific regions of the cDNA of the gamma chain were then compared with the cDNAs for the alpha and beta chains of human fibrinogen.

Amino Acid Sequence↗
PubMed1981

Characterization of a cDNA clone coding for the beta chain of bovine fibrinogen.

Recombinant plasmids containing bovine cDNA have been screened with a radiolabeled cDNA enriched for bovine fibrinogen. A number of plasmids containing cDNAs for fibrinogen were identified by this assay. One plasmid, designated pBI beta 1, was found to contain a cDNA insert of 1372 base pairs. The sequence of the cDNA insert for this plasmid was then determined. It was shown to code for 424 amino acids of the beta chain of fibrinogen, starting with residue 44. This and other data made it possible to construct the complete amino acid sequence of the beta chain of the protein. Comparison of the amino acid sequence of the beta chain of bovine fibrinogen with the corresponding chain of the human molecule indicated that the two chains are greater than 80% homologous.

Amino Acid Sequence↗
PubMed1980

The biosynthesis of bovine fibrinogen, prothrombin, and albumin in a cell-free system.

Preparative amounts of poly A-containing mRNA were isolated from bovine liver. In the presence of a cell-free system from rabbit reticulocytes, this mRNA stimulated the synthesis of a number of plasma proteins including albumin, fibrinogen, prothrombin, and antithrombin III. Automatic Edman degradation of immunoprecipitated albumin showed that this protein was synthesized as preproalbumin containing an NH2-terminal extension of 18 amino acid residues. Prothrombin is also synthesized as a precursor (preprothrombin), and the signal sequence for this protein is also rich in hydrophobic amino acids. The three chains of fibrinogen are synthesized from individual mRNAs, which are then linked by disulfide bonds to form the mature protein.

Amino Acid Sequence↗