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.     

Human platelet glycoprotein IX: an adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures.

The glycoprotein (GP) Ib-IX complex on the surface of human platelets functions as the von Willebrand factor receptor and mediates von Willebrand factor-dependent platelet adhesion to blood vessels. GPIX is a relatively small (Mr, 17,000) protein that may provide for membrane insertion and orientation of the larger component of the complex, GPIb (Mr, 165,000). Using antibody screening, we cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage lambda gt11. Lacking a 5' untranslated region and start codon, the cDNA sequence includes 604 nucleotides, beginning with 495 bases at the 5' end coding for 165 amino acids, followed by a stop codon and 106 noncoding bases at the 3' end. By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A)+ RNA. Translation of the cDNA sequence gives a predicted protein sequence beginning with a truncated putative signal sequence of 5 amino acid followed by a sequence of 17 amino acids matching that determined directly by Edman degradation of intact GPIX. The predicted amino acid sequence of mature GPIX includes an NH2-terminal extracytoplasmic domain of 134 residues, a transmembrane domain of 20 residues, 6 intracytoplasmic residues, and 1 N-linked glycosylation site. GPIX contains a leucine-rich glycoprotein (LRG) sequence of 24 amino acids similar to conserved LRG sequences in GPIb and other proteins from humans, Drosophila, and yeast. "Flanking" sequences of approximately 22 amino acids are present at the NH2 and/or COOH sides of the "central" LRG sequence(s) in GPIX, GPIb, and the other human and Drosophila members of the LRG family. The role of the flank-LRG center-flank structure in the evolution and function of the LRG proteins remains to be defined.     

Cloning and characterization of human liver cDNA encoding a protein S precursor.

Human liver cDNA encoding a protein S precursor was isolated from two cDNA libraries by two different techniques. Based upon the frequency of positive clones, the abundance of mRNA for protein S is approximately 0.01%. Blot hybridization of electrophoretically fractionated poly(A)+ RNA revealed a major mRNA approximately 4 kilobases long and two minor forms of approximately 3.1 and approximately equal to 2.6 kilobases. One of the cDNA clones contains a segment encoding a 676 amino acid protein S precursor, as well as 108 and 1132 nucleotides of 5' and 3' noncoding sequence, respectively, plus a poly(A) region at the 3' end. The cDNAs are adenosine plus thymidine-rich (60%) except for the 5' noncoding region, where 78% of the nucleotides are guanosine or cytosine. The protein precursor consists of a 41 amino acid "leader" peptide followed by 635 amino acids corresponding to mature protein S. Comparison of the mature protein region with homologous vitamin K-dependent plasma proteins shows that it is composed of the following domains: an amino-terminal gamma-carboxyglutamic acid-rich region of 37 amino acids; a 36 amino acid linker region rich in hydroxy amino acids; four epidermal growth factor-like segments, each approximately 45 amino acids long; and a 387 amino acid carboxyl-terminal domain of unrecognized structure and unknown function.     

Cloning and sequence of cDNA coding for alpha 1-antitrypsin.

Recombinant plasmids containing human and baboon cDNA have been screened for alpha 1-antitrypsin, a major serine protease inhibitor present in blood. One plasmid, designated pBa alpha 1a2, was found to contain a cDNA insert of 1352 base pairs coding for the baboon inhibitor. It included 45 nucleotides that code for 15 amino acids present in the amino-terminal signal sequence of the protein, 1182 nucleotides that code for 394 amino acids in the mature protein, a stop codon, and a noncoding region of 76 nucleotides. Comparison of the amino acid sequences of baboon alpha 1-antitrypsin, human antithrombin III, and chicken ovalbumin indicated that these three proteins are about 230% homologous. A second plasmid, designated pH alpha 1a1, was found to contain a human cDNA insert of 306 base pairs. This plasmid coded for 69 amino acids present in the carboxyl-terminal region of human alpha 1-antitrypsin. The human and baboon cDNAs and their amino acid sequences are greater than 96% homologous.     

Cloning of a cDNA coding for human factor V, a blood coagulation factor homologous to factor VIII and ceruloplasmin.

Coagulation factor V is a high molecular weight plasma glycoprotein that participates as a cofactor in the conversion of prothrombin to thrombin by factor Xa. A phage lambda gt11 Hep G2 cell cDNA expression library was screened by using an affinity-purified antibody to human factor V, and 11 positive clones were isolated and plaque-purified. The clone containing the largest cDNA insert contained 2970 nucleotides and coded for 938 amino acids, a stop codon, and 155 nucleotides of 3' noncoding sequence including a poly(A) tail. The coding region includes 651 amino acids from the carboxyl terminus that constitute the light chain of human factor Va and 287 amino acids that are part of the connecting region of the protein. The predicted amino acid sequence agreed completely with 147 amino acid residues that were identified by Edman degradation of cyanogen bromide peptides isolated from the light chain. During the activation of factor V, several peptide bonds are cleaved by thrombin, giving rise to a heavy chain, a connecting fragment(s), and a light chain. The light chain is generated by the cleavage of an Arg-Ser peptide bond. The amino acid sequence of the light chain is homologous (40%) with the carboxyl-terminal fragment (Mr, 73,000) of human factor VIII. Both fragments have a similar domain structure that includes a single ceruloplasmin-related domain followed by two C domains. The carboxyl terminus of the connecting region, however, shows no significant amino acid sequence homology with factor VIII. It is very acidic and contains a number of potential N-linked glycosylation sites. It also contains about 20 tandem repeats of nine amino acids.     

GTPase of bovine rod outer segments: the amino acid sequence of the alpha subunit as derived from the cDNA sequence.

The sequence of the 350 amino acids in the alpha subunit of GTPase of bovine rod outer segments has been determined. Enriched GTPase mRNA was used to prepare a cDNA library in the expression vector lambda gt11 and several overlapping cDNA clones corresponding to the alpha subunit of the GTPase were identified. The cDNA sequence determined contains 93 nucleotides upstream of the 5' end of the coding region, 1050 nucleotides that specify the amino acid sequence, and 45 nucleotides downstream from the 3' end. The previously described partial amino acid sequences and the sequences at the ADP-ribosylation sites for cholera and pertussis toxins are all confirmed and fitted into the present complete sequence. Homologies are found between the sequence of the alpha subunit and those of other guanine nucleotide-binding proteins, the ras proteins, peptide chain elongation factors EF-Tu and EF-G, and the initiation factor IF2.     

Molecular cloning and sequencing of cDNAs encoding the entire rat fatty acid synthase.

Overlapping cloned cDNAs representing the entire sequence of the rat fatty acid synthase mRNA have been isolated from a cDNA library and sequenced. Authenticity of the cDNA clones was supported by hybridization to fatty acid synthase mRNA and by amino-terminal sequencing of 39 fatty acid synthase CNBr fragments. The full-length fatty acid synthase mRNA is 9156 nucleotides long and includes an 84-nucleotide 5' noncoding region, a 7515-nucleotide coding sequence, and a 1537-nucleotide 3' noncoding region; a second mRNA species containing a shortened 3' noncoding sequence is also transcribed in the rat. The encoded fatty acid synthase subunit contains 2505 amino acids and has a molecular weight of 272,340. Active sites and substrate binding sites were located within the sequence, thus establishing the order of domains on the multifunctional animal fatty acid synthase as condensing enzyme-transferase-dehydrase-enoyl reductase-ketoreductase-acyl carrier protein-thioesterase.     

Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone.

We have cloned cDNA copies of human preproparathyroid hormone in Escherichia coli after insertion of double-stranded DNA into the Pst I site of plasmid pBR322 using the poly(dG) . poly(dC) homopolymer extension technique. Recombinant plasmids coding for preproparathyroid hormone were identified by filter hybridization assay using as a probe 32P-labeled bovine preproparathyroid cDNA. Nucleotide sequence analysis of five recombinant plasmids permitted the assignment of 74 nucleotides of the 5' noncoding region, the entire coding region of 345 nucleotides, and the entire 3' noncoding region of 348 nucleotides of the mRNA. The coding sequence predicts the previously unknown "pre" amino acid sequence and clarifies the hormone's amino acid sequence, which has been disrupted. The 5' noncoding region contains an AUG codon followed by a UGA stop codon before the authentic initiator codon. The 3' noncoding region is 120 nucleotides longer than in bovine preproparathyroid mRNA and contains two A-A-U-A-A-A sequences, potential signals for polyadenylation.     

Characterization of the mRNA and cloned cDNA specifying the third component of mouse complement.

Eighteen cDNA clones containing inserts specific for the third component of complement (C3) have been derived from high molecular weight mouse liver mRNA. The inserts span 4,600 nucleotides of the C3 coding sequence, including the 3' end of C3 mRNA. The length of C3 mRNA was determined to be 5,100 +/- 200 nucleotides, including a poly(A)-containing tail of mean length 170 nucleotides. From cDNA sequence analysis of the 5'-proximal region of C3 mRNA, the NH2-terminal amino acid sequence of the mature C3 beta chain was predicted to be Ile-Pro-Met-Tyr-Ser-Ile-Ile-Thr-Pro-Asn-Val-Leu-Arg-Leu-Glu. This sequence is in good agreement with the reported amino acid sequences of human and guinea pig C3 beta chains. These data position the C3 beta subunit to the NH2-terminal portion of the precursor C3 molecule (pro-C3) and establish the order of subunits in pro-C3 to be NH2-beta-alpha-COOH. In addition, the cDNA sequence indicates that an NH2-terminal extension peptide precedes the beta chain in pro-C3. The amino acid sequence of the mouse C3a fragment and its flanking regions was determined. The data indicate the presence of four arginine residues located between the COOH terminus of the C3 beta and the NH2 terminus of the C3 alpha subunits in pro-C3. The coding sequences of the amino acids that constitute the internal thioester domain in C3 were determined. Unexpectedly, the glutamyl residue that has been shown to participate in the thioester bond in native C3 was found to be encoded as a glutamine.     

The nucleotide sequence of the gene for human protein C.

A human genomic DNA library was screened for the gene for protein C by using a cDNA probe coding for the human protein. Three different overlapping lambda Charon 4A phage were isolated that contain inserts for the gene for protein C. The complete sequence of the gene was determined by the dideoxy method and shown to span about 11 kilobases of DNA. The coding and 3' noncoding portion of the gene consists of eight exons and seven introns. The eight exons code for a preproleader sequence of 42 amino acids, a light chain of 155 amino acids, a connecting dipeptide of Lys-Arg, and a heavy chain of 262 amino acids. The preproleader sequence and the connecting dipeptide are removed during processing, resulting in the mature protein composed of a heavy and a light chain held together by a disulfide bond. The heavy chain also contains the catalytic region for the serine protease. Two Alu sequences and two homologous repeats of about 160 nucleotides were found in intron E. The seven introns in the gene for protein C are located in essentially the same positions in the amino acid sequence as the seven introns in the gene for human factor IX, while the first three introns in protein C are located in the same positions as the first three in the gene for human prothrombin.     

Amino acid sequence of the von Willebrand factor-binding domain of platelet membrane glycoprotein Ib.

We report the amino acid sequence of a 299-residue segment from the alpha chain of the human platelet membrane glycoprotein Ib. This includes the complete sequence of the amino-terminal tryptic fragment of 290 residues comprising the von Willebrand factor-binding domain. Two primary sets of overlapping fragments were obtained by cleavage of the S-carboxymethylated protein at methionyl and lysyl bonds following treatment with cyanogen bromide and Achromobacter protease I, respectively. Additional fragments were obtained by treatment of native glycocalicin with trypsin, Staphylococcus aureus V8 protease, and Serratia marcescens protease. Analysis of all these fragments provided data that allowed determination of the continuous sequence corresponding to approximately half of the alpha-chain polypeptide. This region of glycoprotein Ib is largely hydrophobic and contains only two N-linked and one O-linked carbohydrate chains. A hydrophilic region exists between residues 215 and 299, which contains a cluster of 10 negatively charged residues at 269-287. This area is likely to attract positively charged molecules. The hydrophilic, highly glycosylated (at serine and threonine residues) region corresponding to the previously described "macroglycopeptide" and representing the carboxyl-terminal half of the alpha chain is likely to begin at residue 292. The determined sequence of the alpha chain of glycoprotein Ib contains a region (residues 29-193) with seven repeats, which is indicative of gene duplication and is highly homologous to human leucine-rich alpha 2-glycoprotein. This protein sequence agrees completely with that deduced from the cDNA sequence reported by Lopez et al. [Lopez, J.A., Chung, D.W., Fujikawa, K., Hagen, F.S., Papayannopoulou, T. & Roth, G.J. (1987) Proc. Natl. Acad. Sci. USA 84, 5615-5619].     

Cloning of cDNA coding for the beta chain of human complement component C4b-binding protein: sequence homology with the alpha chain.

The major form of complement component C4b-binding protein, a regulator of the complement system, is composed of seven identical 70-kDa alpha chains, each containing a binding site for the complement protein C4b. We recently showed that C4b-binding protein also contains a unique 45-kDa beta chain. It is disulfide-linked to the central core and contains a binding site for the vitamin K-dependent protein S. We have now isolated and characterized full-length cDNA clones for the beta chain. In addition, 57% of the structure was determined by protein sequencing of tryptic and chymotryptic peptides. Two clones, A8 and C1, isolated from different libraries were sequenced. Except for a deleted triplet encoding Ala-3 in clone A8, the two clones were identical and coded for a leader sequence of 17 amino acids and a mature protein of 235 amino acids (including Ala-3). By N-terminal amino acid sequencing, the Ala-3 heterogeneity was confirmed and a third beta-chain species starting at Glu-4 was identified. The beta chain contains five potential N-linked glycosylation sites, and endoglycosidase digestion suggested that the beta chain contained multiple complex carbohydrate side chains. Northern blot analysis of human liver mRNA, using the beta-chain cDNA as the probe, demonstrated a major mRNA species of approximately 1.0 kilobase. From the N terminus, the beta chain contains three tandem repeat units (60 amino acids long) that are homologous to those present in the alpha chain. The C-terminal region, which was unrelated to the tandem repeats, demonstrated sequence similarity with the corresponding region of the alpha chain. In both alpha and beta chains these regions contain two cysteine residues that probably form the interchain disulfide bridges.     

cDNA clone for the heavy chain of the human B cell alloantigen DC1: strong sequence homology to the HLA-DR heavy chain.

A cDNA library has been constructed from a B cell mRNA fraction enriched for HLA-DR sequences, and cDNA clones corresponding to sequences specifically expressed in B lymphocytes have been isolated by a differential screening procedure. Analysis of these clones with probes specific for the HLA-DR heavy chain gene allowed the characterization of HLA-DR heavy chain-related sequences. One clone, pDCH1, was demonstrated to encode the DC1 heavy chain because the amino acid sequence predicted from its nucleotide sequence matches eight out of nine residues available for comparison in the amino-terminal sequence of the DC1 heavy chain. The heavy chain of the DC1 alloantigen is composed of 232 amino acids and can be divided into two external domains, alpha 1 (amino acids 1-87) and alpha 2 (amino acids 88-181), a connecting peptide (amino acids 182-194), a hydrophobic transmembrane region (amino acids 195-217), and an intracytoplasmic region (amino acids 218-232). Comparison with the HLA-DR heavy chain reveals strong sequence homology in the second external Ig-like domain (alpha 2) and the transmembrane region. In contrast, the first external domain, the connecting peptide, and the intracytoplasmic region are less conserved.     

The largest variant of platelet glycoprotein Ib alpha has four tandem repeats of 13 amino acids in the macroglycopeptide region and a genetic linkage with methionine145

Platelet membrane glycoprotein Ib alpha (GPIb alpha) bears the human platelet alloantigen (HPA)-2 and molecular weight (MW) polymorphisms on sodium dodecyl sulfate-polyacrylamide gels. HPA-2 arises from a threonine/methionine dimorphism at residue 145 of the GPIb alpha sequence, whereas different numbers of tandem repeats of a 39-bp sequence encoding 13-amino acids corresponding to a region between serine399 and threonine411 of the GPIb alpha account for the latter. To identify the genetic basis of the MW polymorphism among Japanese, we counted the tandem repeats in 103 individuals. In addition to the reported three variants with one, two, or three tandem repeats, we identified a new variant with four perfect tandem repeats of the 39-bp sequence that corresponded to the largest phenotype. Phenotypic analysis of the MW polymorphism on 12 individuals including all four phenotypes completely accorded in the genotype. We also determined the genotype of HPA-2 and found that methionine145 was in complete linkage disequilibrium, with the larger variants containing three or four tandem repeats. These results imply a model of evolutionary steps in the gene encoding GPIb alpha.     

Sequence of a cDNA clone encoding the polysialic acid-rich and cytoplasmic domains of the neural cell adhesion molecule N-CAM.

Purified fractions of the neural cell-adhesion molecule N-CAM from embryonic chicken brain contain two similar polypeptides (Mr, 160,000 and 130,000), each containing an amino-terminal external binding region, a carbohydrate-rich central region, and a carboxyl-terminal region that is associated with the cell. Previous studies indicate that the two polypeptides arise by alternative splicing of mRNAs transcribed from a single gene. We report here the 3556-nucleotide sequence of a cDNA clone (pEC208) that encodes 964 amino acids from the carbohydrate and cell-associated domains of the larger N-CAM polypeptide followed by 664 nucleotides of 3' untranslated sequence. The predicted protein sequence contains attachment sites for polysialic acid-containing oligosaccharides, four tandem homologous regions of polypeptide resembling those seen in the immunoglobulin superfamily, and a single hydrophobic sequence that appears to be the membrane-spanning segment. The cytoplasmic domain carboxyl terminal to this segment includes a block of approximately equal to 250 amino acids present in the larger but not in the smaller N-CAM polypeptide. We designate these the ld (large domain) polypeptide and the sd (small domain) polypeptide. The intracellular domains of the ld and sd polypeptides are likely to be critical for cell-surface modulation of N-CAM by interacting in a differential fashion with other intrinsic proteins or with the cytoskeleton.     

Extensive intragenic sequence homology in two distinct rat lens gamma-crystallin cDNAs suggests duplications of a primordial gene.

The nucleotide sequences of two different rat lens gamma-crystallin cDNA clones, pRL gamma 2 and pRL gamma 3, have been determined. pRL gamma 3 contains the complete coding information for a gamma-crystallin of 173 amino acids whereas pRL gamma 2 is incomplete in that it lacks the codons for the first three amino acids of a separate but very homologous gamma-crystallin of identical length. Both rat gamma-crystallins are homologous to the known amino acid sequence of bovine gamma-crystallin II which is only a single amino acid longer. The length of the region downstream the coding sequence to the A-A-T-A-A-A polyadenylylation signal sequence is 40 nucleotides in each clone. In pRL gamma 3 the poly(A) signal sequence is followed at 14 nucleotides by a remnant of the poly(A) tail which indicates that this clone contains a complete 3' noncoding region. pRL gamma 2 has only seven nucleotides following this signal sequence and no poly(A) tail, suggesting an incomplete 3' end. The cDNA clones show an overall nucleotide sequence homology of 85%. The mutual homology at the amino acid level is 73% whereas their amino acid homology with bovine gamma-crystallin II is about 70%. The nucleotide sequence of each clone also reveals a high intragenic homology and seems to be duplicated in itself. We suggest that the gamma-crystallin genes have arisen by multiple duplications of a primordial gene which consisted of about 120 nucleotides.     

Complete primary structures of two major murine serum amyloid A proteins deduced from cDNA sequences.

cDNA clones encoding two major mouse serum amyloid A proteins, SAA1 and SAA2, were isolated from a liver cDNA library of the lipopolysaccharide-stimulated BALB/c mouse, and their nucleotide sequences were determined. The insert of the SAA2 cDNA clone contained 607 nucleotides with a 5' untranslated region of 36 nucleotides, a signal peptide region corresponding to 19 amino acids, a mature protein region corresponding to 103 amino acids, and a 3' untranslated region of 202 nucleotides. The SAA1 cDNA insert contained 549 nucleotides specifying a part of a signal peptide region, a mature protein region, and a 3' untranslated region. A comparison of the nucleotide and deduced amino acid sequences of SAA1 cDNA with that of SAA2 cDNA showed a high degree of homology: 95% nucleotide sequence homology in the coding region (91% amino acid sequence homology) and 90% homology in the 3' untranslated region. One of nine amino acid differences between SAA1 and SAA2 predicted from the cDNA sequences was located in a putative proteolytic cleavage site for amyloid A protein formation: SAA2 had the Thr-Met sequence in this site, while SAA1 had the Thr-Ile sequence. This suggests that SAA1, which does not deposit as amyloid A protein, is also potentially susceptible to putative proteolytic enzymes. In addition, as compared with mouse SAA2, human SAA1, monkey and mink amyloid A protein, mouse SAA1 had two unique substitutions, which may play a role in differential deposition of mouse SAA isotypes in amyloid tissues.     

Mutation of leucine-57 to phenylalanine in a platelet glycoprotein Ib alpha leucine tandem repeat occurring in patients with an autosomal dominant variant of Bernard-Soulier disease.

The primary sequences of the three individual glycoprotein (GP) chains, GPIb alpha, GPIb beta, and GPIX, comprising the normal platelet GPIb/IX receptor for von Willebrand factor (vWF) have recently been determined, opening the possibility for characterization of disorders of this receptor at the molecular level. The presence of a leucine tandem repeat in each of these chains is of particular interest, because such repeats may be involved in associations between polypeptide segments. We now report an autosomal dominant variant of Bernard-Soulier disease associated with the heterozygous substitution of phenylalanine for a highly conserved leucine residue within the GPIb alpha leucine tandem repeat. Affected individuals experienced a moderate bleeding tendency, thrombocytopenia, and an increased mean platelet volume. Platelet aggregation was decreased only in response to ristocetin or to asialo-vWF. The kd for 125I-vWF binding to patients platelets was significantly increased over control values at 0.5 mg/mL ristocetin, but was normal at 1.0 or 1.5 mg/mL ristocetin. While sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an essentially normal complement of all components of the GPIb/IX complex, a minor amount of a putative proteolytic fragment was identified that migrated faster than GPIb and was immunoreactive with polyclonal anti-GPIb alpha antibody, but not with a monoclonal antibody directed against the 45-Kd amino-terminal region of GPIb alpha. However, because the great majority of patient GPIb alpha comigrates with normal GPIb alpha, the major functional abnormalities of the patient platelets are most likely a consequence of the altered structure of the nonproteolyzed protein. Full concordance within the studied family between phenotypic expression and a heterozygous single nucleotide substitution in genomic DNA coding for a phenylalanine in place of the wild-type leucine at residue 57 of the mature GPIb alpha, absence of this substitution in 266 alleles from the normal population, and the lack of any other abnormality of patient DNA throughout the entire coding sequence for GPIb alpha provide strong support that this substitution may constitute a pathologic point mutation responsible for the observed phenotypic abnormalities. While the roles that leucine tandem repeats may normally play within the GPIb/IX complex are not yet known, the perturbation of such a repeat in GPIb alpha may impair interaction with other components of the complex and/or with the binding of vWF.     

Molecular cloning of the cDNA and chromosomal gene for vitamin D-dependent calcium-binding protein of rat intestine.

A cDNA encoding the vitamin D-dependent rat intestinal calcium-binding protein has been isolated by screening a rat intestinal cDNA library. The cDNA is 406 nucleotides long and appears to contain all the sequences of the mRNA. The cDNA includes the entire protein coding region. It consists of 237 nucleotides coding for 79 amino acids, including the starting methionine, flanked by 62 and 107 noncoding nucleotides at the 5' and 3' ends, respectively. Using the cloned cDNA, we have isolated a genomic clone from a rat liver genomic library. Restriction mapping and Southern analysis using synthetic oligonucleotides localized the gene to a 4.0-kilobase-pair HindIII fragment.