Partial amino acid sequence of human thrombospondin as determined by analysis of cDNA clones: homology to malarial circumsporozoite proteins

A lambda gt 11 library prepared from human umbilical vein endothelial cell RNA was screened for cDNAs encoding thrombospondin. Reagents included a monospecific antibody to human thrombospondin and a mixture of four synthetic oligodeoxyribonucleotides derived from an amino acid sequence near the NH2 terminus of mature human thrombospondin. Two series of cDNA clones coding for sequences at the 5' and 3' ends of thrombospondin mRNA, respectively, were isolated. The nucleotide sequence of a 1.3-kilobase (kb) 5' clone (lambda TS-33) coded for 99 bases of 5' untranslated RNA, a signal peptide of 18 amino acids, and the first 379 amino acids of thrombospondin. Northern blot analysis with lambda TS-33 detected a single mRNA species of approximately 6.0 kb in rat aortic smooth muscle cell RNA. Thrombospondin mRNA levels increased rapidly, but transiently, in quiescent smooth muscle cells treated with platelet-derived growth factor. The kinetics of this response were very similar to those of the thrombospondin protein to this growth factor. There was significant homology in amino acid sequence between thrombospondin and a conserved region in the circumsporozoite protein of two malarial sporozoites. This region of thrombospondin may therefore represent a potential recognition site for a cell surface thrombospondin receptor.     

Molecular cloning of the uteroferrin-associated protein, a major progesterone-induced serpin secreted by the porcine uterus, and the expression of its mRNA during pregnancy

The uteroferrin(Uf)-associated basic proteins (UfAP) are a group of three (Mr = 42K, 48K, and 50K) antigenically related, basic glycoproteins secreted by the porcine uterus under the influence of progesterone (P4) which exist as heterodimers (Mr = 80,000) with the iron-binding acid phosphatase, Uf. Several UfAP cDNA clones from a day-60 pregnant pig uterine endometrial cDNA library have been cloned and sequenced. The UfAP mRNA is approximately 1400 bases long and has a single open reading frame of 1251 bases with two start codons at positions 64 and 79 from the 5'-end. UfAP mRNA content of the endometrium increases as pregnancy proceeds, reaching maximum levels around day 70 and then remaining relatively constant in late gestation (days 70 to 110). The pro-form of the UfAP minus signal sequence appears to be 392 amino acids in length and has four potential N-linked glycosylation sites Asn107, Asn197, Asn243, and Asn315. Comparison of the NH2-terminal sequences of the individual UfAP poly-peptides with the amino acid sequence deduced from the cDNA has indicated a series of at least four posttranslational proteolytic processing steps which generate the various molecular forms of the UfAP. The deduced amino acid sequence of UfAP shares considerable identity with several protease inhibitors and hormone-binding proteins that are members of the serpin superfamily of proteins. The UfAP amino acid sequence also exhibits about 55% sequence identity with the P4-induced uterine milk proteins (UTMP) of the sheep. Since the UfAP and UTMP share many biosynthetic and structural features that include site of biosynthesis in the endometrium, P4-responsiveness, the presence of the mannose 6-phosphate lysosomal recognition marker, and considerable sequence similarity, the UfAP and the UTMP may have homologous function which for both still remains obscure.     

Structure of rodent helix-destabilizing protein revealed by cDNA cloning

A cDNA library of newborn rat brain poly(A+) RNA in lambda gt 11 was screened with a synthetic oligonucleotide probe corresponding to a five amino acid sequence in the N-terminal region of the calf helix-destabilizing protein, UP1. Six positive phage were isolated after testing 2 X 10(5) recombinants, and each phage was plaque purified. Four of these phage clones were positive with a second oligonucleotide probe corresponding to a 5 amino acid sequence in the C-terminal region of calf UP1; one of the clones positive with both probes was selected for detailed study. This phage, designated lambda HDP-182, contained a 1706-base pair cDNA insert corresponding to an mRNA with a poly(A) sequence at the 3' terminus and a single open reading frame starting 63 bases from the 5' terminus and extending 988 bases. The 3' untranslated region of the mRNA contained 718 bases, including an AAUAAA signal 21 bases from the poly(A) sequence and a 16-residue poly(U) sequence flanked on each side by oligonucleotide repeats. Primer extension analysis of newborn rat brain poly(A+) RNA suggested that the cDNA insert in lambda HDP-182 was full length except for about 35 nucleotide residues missing from the 5' end untranslated region, and Northern blot analysis revealed one relatively abundant mRNA species of approximately the same size as the cDNA insert. The 988-residue open reading frame in the cDNA predicted a 34,215-dalton protein of 320 amino acids. Residues 2 through 196 of this rat protein are identical to the 195-residue sequence of the calf helix-destabilizing protein, UP1. The 124-amino acid sequence in the C-terminal portion of the 34,215-dalton protein is not present in purified calf UP1. This 124-residue sequence has unusual amino acid content in that it is 11% asparagine, 15% serine, and 40% glycine and consists of 16 consecutive oligopeptide repeats. Computer-derived secondary structure predictions for the 34,215-dalton protein revealed two distinct domains consisting of residues 1 through approximately 196 and residues approximately 197 to 320, respectively.     

Isolation and characterization of a cDNA clone encoding rat 5-lipoxygenase

A full-length cDNA clone encoding 5-lipoxygenase, a key enzyme in the formation of leukotrienes, was isolated from a rat basophilic leukemia cell lambda gt11 cDNA library. The 2.5-kilobase (kb) cDNA insert, whose identity was confirmed by hybrid-select translation and DNA sequence analysis, has a 2.0-kb open reading frame encoding a protein of Mr approximately 77,600 and includes 60 base pairs of 5'-untranslated region and 0.4 kb of 3'-untranslated region to the polyadenylation signal. The deduced amino acid sequence shows significant homology with published sequences for the rabbit reticulocyte lipoxygenase and soybean lipoxygenase-1; it also contains sequences similar to a consensus sequence found in several calcium-dependent membrane-binding proteins. The cDNA recognizes a 2.6-kb mRNA species which is detected in all tissues but is particularly abundant in RNA from lung.     

Isolation and characterisation of a cDNA clone for a chlorophyll synthesis enzyme from Euglena gracilis. The chloroplast enzyme hydroxymethylbilane synthase (porphobilinogen deaminase) is synthesised with a very long transit peptide in Euglena

A cDNA expression library was constructed from light-grown Euglena gracilis poly(A)-rich RNA in lambda gt11. Antibodies to Euglena hydroxymethylbilane synthase, the third enzyme in the porphyrin biosynthetic pathway, were used to screen the library and a clone encoding part of the sequence of hydroxymethylbilane synthase was identified. This was used to rescreen the library and a full-length clone was isolated, which encoded not only the entire mature protein (Mr 36,927), but also an N-terminal extension of 139 amino acids. The deduced Mr of the whole polypeptide is 51,744, which corresponds to the size of the protein immunoprecipitated from the translation products of Euglena poly(A)-rich RNA. The mature protein is 60-70% similar to hydroxymethylbilane synthase from human erythrocytes and Escherichia coli. The sequence of the N-terminal extension has similarities to both the transit peptides of chloroplast proteins and those for the endoplasmic reticulum. This is the first report both of a cDNA clone for an enzyme of the chlorophyll biosynthetic pathway and of a putative transit peptide for a nuclear-encoded Euglena protein.     

Mammalian alpha-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots

A new polyclonal antibody against the alpha-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell alpha-polymerase. The antibody neutralized alpha-polymerase activity and was strong and specific for the alpha-polymerase catalytic polypeptide (Mr 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in lambda gt11. A positive phage was identified and plaque purified. This phage, designated lambda pol alpha 1.2, also was found to be positive with an antibody against Drosophila alpha-polymerase. The insert in lambda pol alpha 1.2 (1183 base pairs) contained a poly(A) sequence at the 3' terminus and a short in-phase open reading frame at the 5' terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified alpha-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating alpha-polymerase. This indicated the lambda pol alpha 1.2 insert encoded an alpha-polymerase epitope and suggested that the cDNA corresponded to an alpha-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding alpha-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed that this mRNA is approximately 5.4 kilobases.     

Sequence of human DNA polymerase beta mRNA obtained through cDNA cloning

A cDNA library from polyA+ RNA of a human teratocarcinoma cell line in phage lambda gt11 was screened with a fragment of the rat beta-polymerase cDNA, lambda pol beta-10, as probe. Five positive phage were identified and plaque purified. The cDNA of one positive clone selected for detailed study was 1257 bp. This insert was sequenced and found to contain the coding region for beta-polymerase, as well as 163 bp and 137 bp from the 5' and 3' untranslated regions, respectively. The primary structure of human beta-polymerase (318 amino acids, Mr = 36, 133) deduced from the cDNA was similar to rat beta-polymerase (95% matched residues). The greatest difference between the sequences of the human and rat cDNAs was in the 3' untranslated regions (64% matched base residues). These results provide necessary sequence information for study of the human beta-polymerase gene.     

Nucleotide sequence of cDNA containing the complete coding sequence for human lysosomal glucocerebrosidase

Complementary DNA clones for human glucocerebrosidase were isolated from a human hepatoma library in lambda gt11. The complete nucleotide sequence of the 1805-base pair cDNA insert has been determined. In addition to 5' and 3' untranslated regions (51 and 206 base pairs, respectively), the cDNA insert contains 1548 base pairs that completely encode human glucocerebrosidase. All possible N-linked glycosylation sites are identified. Examination of the 19 amino acids of the leader polypeptide beginning with the ATG at position 52 revealed a hydrophobic core and a carboxyl-terminal glycine at the peptidase cleavage site, features consistent with the leader sequences described for other human translocated proteins. The Mr of 57,000 calculated from the 516 amino acids deduced from cDNA sequence is in good agreement with that identified by immunoprecipitation following in vitro translation of human placental mRNA.     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

Sequence of a HeLa cDNA provides the DNA binding domain and carboxy terminus of HE47: a human helix-loop-helix protein related to the enhancer binding factor E47.

In order to identify cDNA encoding regulators of gene expression, a HeLa lambda gt11 expression library was screened with a DNA segment containing multiple copies of a sequence spanning the AP4 site in the simian virus 40 control region. We identified a partial cDNA encoding HE47, a sequence-specific DNA binding protein. The HeLa clone overlaps with a previously reported human B-cell partial cDNA encoding E47. The HeLa cDNA includes the HE47 DNA binding domain, its carboxy terminus, and the 3' untranslated region of its mRNA.     

Molecular cloning and nucleotide sequence of cDNA encoding hydroxyindole O-methyltransferase of bovine pineal glands

Messenger RNA for hydroxyindole O-methyltransferase (EC 2.1.1.4) was partially purified from poly(A)+ RNA isolated from bovine pineal glands by sucrose density gradient centrifugation. The enriched mRNA was used to prepare a cDNA library by use of expression vector lambda gt11. The library was screened with monoclonal antibodies to the enzyme, and three cDNA clones were isolated. These cloned cDNAs cross-hybridized with one another, and their fusion proteins reacted to the monoclonal antibodies with different binding properties. Hydroxyindole O-methyltransferase enzymatic activity was demonstrated in the bacteria lysate infected with lambda HIOMT-A16, the clone that contained the longest insert. An almost full-length cDNA clone was isolated from lambda gt10 cDNA library by use of the lambda HIOMT-A16 cDNA as a probe. The primary structure of hydroxyindole O-methyltransferase was determined by analyzing the nucleotide sequence of the cDNAs. It consisted of 1939 nucleotides including a 1050-nucleotide region coding for 350 amino acids. RNA transfer blot analysis indicated that mRNA encoding hydroxyindole O-methyltransferase was present only in the pineal gland and not in the brain, retina, and liver of cow.     

Cloning and nucleotide sequence of ovine prolactin cDNA

A cDNA expression library was constructed in the lambda gt 11 phage vector using ovine (o) pituitary mRNA. The clone, pOP1, carrying a 934-bp insert contains an open reading frame beginning with the first nucleotide (nt) and ending with the stop codon TAA at nt position 781. Two potential translation start codons (ATGs) are present in the 5' region of this cDNA. Translation initiation could occur at the 5' proximal ATG at nt position 61. The nucleotide sequence around this ATG (TCCATGG), resembles the optimum sequence context for translation initiation by the eukaryotic ribosomes, as defined by mutational analysis [Kozak, Cell 44 (1986) 283-292)], with its substitution of the A at -3 of the consensus sequence by a T residue in this clone. Translation initiated at this codon could potentially code for the entire pre-prolactin (pre-PRL) molecule. The 3'-untranslated region is 154 nt long and contains a polyadenylation signal AATAAA. The deduced amino acid sequence agrees in totality with the published amino acid sequence of the mature hormone. The present study reports on the nucleotide sequence of o-PRL mRNA and the deduced amino acid sequence in the signal peptide of the hormone.