Structure and expression of a cloned cDNA for mouse interferon-beta

A unique sequence in the mouse genome which cross-hybridized to a cloned human interferon-beta 1 gene was detected by DNA blot analysis. Taking advantage of this, a cDNA library prepared from partially purified mRNA for mouse interferon-beta was screened using human interferon-beta 1 DNA as a probe. One of the positive clones, pM beta-3, contained a 680-base pair cDNA insert, whose base sequence contained a single large open reading frame for 182 amino acids. The coding sequences of the cDNA showed homologies of 63% at the nucleotide and 48% at the amino acid level with respect to human interferon-beta 1 cDNA (Taniguchi, T., Ohno, S., Fujii-Kuriyama, Y., and Muramatsu, M. (1980) Gene 10, 11-15). The first 21 amino acids, considered to be the signal peptide, were followed by 24 amino acids, whose sequence was identical with the NH2-terminal sequence that had been reported for mouse interferon-beta from Ehrlich ascites tumor cells (Taira, H., Broeze, R. J., Jayaram, B. M., Lengyel, P., Hunkapiller, M. W., and Hood, L. E. (1980) Science (Wash. D.C.) 207, 528-530). The complete primary sequence of mature interferon-beta polypeptide consisting of 161 amino acids (Mr = 19,700) was deduced. There are three N-glycosylation sites, and this offers an explanation for the larger molecular size (Mr = 26,000-40,000) of natural mouse interferon-beta in comparison to the deduced interferon polypeptide. The cDNA, when fused to a SV40 promoter sequence and then introduced into COS-7 cells, directed the synthesis and secretion of a protein product indistinguishable from the authentic mouse interferon-beta.     

Nucleotide sequence of cloned cDNA coding for mouse epsilon casein

We isolated cDNA clones, which correspond to the mRNA coding for the smallest of the seven mouse caseins. From the nucleotide sequence of the cDNA we deduced the amino acid sequence of the protein, which we named epsilon casein. Mouse epsilon casein and cow alpha s2 casein show amino acid sequence homologies in the N-terminal region of the mature protein. The signal peptide of mouse epsilon casein shows in length and sequence remarkable homology to the signal peptides of the calcium-precipitable caseins of other species. In accordance with this group of caseins mouse epsilon casein contains in the sequence -Ser-Ser-Glu-Glu- a site for potential multiple phosphorylation.     

Nucleotide sequence of a mouse kappa light chain cDNA cloned in a bacterial plasmid.

A mouse MOPC21 cDNA previously cloned in plasmid pMB9(Higuchi $\text{et}\text{al.}$, Proc. Natl. Acad. Sci. 73 (1976) 2136–2140; Wall $\text{et}\text{al.}$, Nucleic Acid Res. 5 (1978) 3113–3128) and is designated pL21-3 has been extensively characterized. Cleavage of pL21-3 with Hpall has shown the insert to be 910 basepairs long, consistent with the length of the entire variable and constant regions and the untranslated regions. Digestion of pL21-3 with various restriction endonucleases has established that the insert sequence starts from parts of the 5′ leader region and extends downstream to include the untranslated 3′ terminus. 131 nucleotides in the variable region corresponding to amino acids 49–91 have been determined.     

Nucleotide sequence of a cloned cDNA corresponding to secreted mu chain of mouse immunoglobulin

The cDNA complementary to a mouse immunoglobulin mu heavy chain mRNA has been cloned into the PstI site of the plasmid vector pBR322. A hybrid plasmid pmu 183.5 containing a 1850 bp insert has been selected by differential screening. The nucleotide sequence of the inset encodes the four constant domains, the terminal piece and the 3'-untranslated region.     

Complete nucleotide sequence of a cloned chicken alpha-globin cDNA.

Chicken globin double-stranded cDNA was synthesised from anaemic adult reticulocyte alpha- and beta-globin mRNA and ligated into the Hind III site of pBR322 using synthetic Hind III decamers. Transformation of E. coli x1776 resulted in the production of a number of alpha- and beta-cDNA clones. One of the alpha-type clones (pCG alpha-8) was fully sequenced and found to code for neither alpha A- nor alpha D-globin. Partial sequencing of the other alpha-cDNA clones indicates that they are all of the same type.     

The nucleotide sequence of a cloned human leukocyte interferon cDNA

We have determined the nucleotide sequence of the human leukocyte interferon cDNA carried in hybrid plasmid Z-pBR322(Pst)/HcIF-2h, which has been shown to direct the formation of a polypeptide with human leukocyte interferon activity (Nagata et al., 1980). The 910 base pair insert contains a 567 (or 543) base pair coding sequence, which determines a putative preinterferon polypeptide consisting of a signal peptide of 23 (or less likely 15) amino acids, followed by an interferon polypeptide of 166 amino acids (calculated molecular weight, 19 390). The coding sequence is preceded by a (most likely incomplete) 56 bp leader and followed by a 242 bp trailer and seven A residues from the poly(A) tail: A comparison of the sequence of 35 amino terminal amino acids of lymphoblastoid interferon (Zoon et al., 1980; M. Hunkapiller and L. Hood, personal communication) and the corresponding sequence deducted for leukocyte interferon revealed 9 differences. This suggests that these two interferons are encoded by two non-allelic genes.     

Human alpha-L-fucosidase: complete coding sequence from cDNA clones

The human lysosomal storage disorder fucosidosis results from the deficiency of alpha-L-fucosidase, a lysosomal enzyme essential for the catabolism of oligosaccharides containing alpha-L-fucosides. cDNA clones coding for human alpha-L-fucosidase have been isolated from lambda gt10 and lambda gt11 cDNA libraries derived from human liver, placenta and colon. Compilation of cDNA sequences results in a nucleotide sequence of 2053 base pairs encoding alpha-L-fucosidase. The sequence contains an open reading frame of 461 amino acids beginning with the first in-frame methionine and includes 439 amino acids which comprise the mature protein in addition to a hydrophobic signal peptide sequence of 22 amino acids.     

Nucleotide sequence of cloned cDNA coding for preproricin

The primary structure of a precursor protein that contains the toxic (A) and galactose-binding (B) chains of the castor bean lectin, ricin, has been deduced from the nucleotide sequence of cloned DNA complementary to preproricin mRNA. A cDNA library was constructed using maturing castor bean endosperm poly(A)-rich RNA enriched for lectin precursor mRNA by size fractionation. Clones containing lectin mRNA sequences were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing all possible sequences for a peptide of the ricin B chain. The entire coding sequence of preproricin was deduced from two overlapping cDNA clones having inserts of 1614 and 1049 base pairs. The coding region (1695 base pairs) consists of a 24-amino-acid N-terminal signal sequence (molecular mass 2836 Da) preceding the A chain 267 amino acids, molecular mass 29 399 Da), which is joined to the B chain (262 amino acids, molecular mass 28 517) by a 12-amino-acid linking region (molecular mass 1385 Da).     

Nucleotide sequence of calf prorennin cDNA cloned in Escherichia coli

The nucleotide sequence of prorennin (prochymosin) cDNA cloned in E. coli was determined by the technique of Maxam and Gilbert. The longest prorennin cDNA insert in pTACR1 contained the putative signal sequence and the coding sequence for the peptide from the 1st amino acid, Ala (NH2 terminal), to the 296th, Ser, and the other clone pTACR9 contained the coding sequence from the 258th, Asp, to the 365th, Ile (COOH terminal), and the TGA termination codon followed by the 3'-untranslated region. Thus, the whole coding sequence for prorennin was obtained in the pair of pTACR1 and pTACR9.     

Nucleotide sequence of cloned cDNA for human pancreatic kallikrein

Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed.     

Nucleotide sequence analysis of the cloned salmon preproinsulin cDNA

A cDNA library was constructed using polyadenylated RNA from salmon (Oncorhynchus keta) Brockmann bodies, plasmid vector pBR322, and in vitro recombinant DNA techniques. Insulin-related clones were identified with a cDNA probe generated from the same RNA and enriched for insulin sequences. Two recombinants were shown to contain the nucleotide sequence of the entire coding region and parts of the 5' and 3' untranslated regions. The salmon preproinsulin mRNA is about 760 nucleotides long, 315 of which code for the protein, while about 190 and 200 nucleotides belong to the 5' and 3' flanking regions, respectively. Comparison of the nucleotide sequences of salmon insulin mRNA with those from other species reveals that sequence conservation is limited to the regions coding for the B and A peptides and two segments of the signal peptide. The C-peptide region exhibits no significant sequence homology with the C-peptides of other vertebrates. The 5' and 3' untranslated regions of the salmon preproinsulin mRNA are homologous only with the anglerfish mRNA, whereas there is no evident homology with those of birds and mammals. In addition to establishing the sequence of the preproinsulin mRNA, cloned salmon insulin cDNA provides a specific probe for the analysis and isolation of genomic DNA fragments containing insulin genes.     

Nucleotide sequence of mouse Tcp-1a cDNA

We have isolated complete cDNA clones encoding the mouse t-complex polypeptides 1A and 1B (TCP-1A and TCP-1B) from t-haplotype and wild-type (wt) mice, respectively. The complete nucleotide (nt) sequence of the Tcp-1a cDNA was determined. The Tcp-1a cDNA has an open reading frame (ORF) encoding a 60-kDa protein of 556 amino acids (aa). A comparison of nt sequences between the Tcp-1a and Tcp-1b cDNAs revealed that the 1786-bp regions upstream from their polyadenylation signals differed by 17 substitutions and that Tcp-1a had different polyadenylation sites from Tcp-1b. In these ORFs, 15 bp were substituted between the two alleles, occurring in 14 codons and resulting in eleven single-aa substitutions. Among these 15 substitutions, twelve were nonsynonymous (aa change) and three were synonymous (no aa change). The aa substitution in TCP-1 has occurred at least 20 times faster between t-haplotype and wt than between mouse and human or mouse and Drosophila.     

The sequence of human parathymosin deduced from a cloned human kidney cDNA

The amino acid sequence of human parathymosin has been deduced from the cDNA sequence of a clone isolated from a human kidney cDNA library. Screening of the cDNA library with a probe containing a partial rat cDNA sequence yielded two clones containing inserts of 1200 and 1100 base pairs respectively, each including the complete open reading frame for human parathymosin. The open reading frame contains 306 nucleotides, including the codon for the initiator methionine. Analysis of the 5' flanking sequence excluded the presence of a hydrophobic signal peptide in the translated sequence. It may therefore be concluded that parathymosin, like prothymosin alpha, is synthesized without formation of a large precursor polypeptide. Comparison of the deduced amino acid sequence with the known primary structure of rat and bovine parathymosins shows that the primary structure of parathymosin is highly conserved among these species.     

The nucleotide sequence of the mouse embryonic beta-like y-globin messenger RNA as determined from cloned cDNA

We have determined the nucleotide sequence of two cloned cDNAs corresponding to the mRNA of mouse embryonic y2 globin. The combined overlapping sequences span a total of 480 bp, beginning at the codon corresponding to amino acido residue 21 and extending to the AATAAA sequence in the 3' untranslated region. Therefore, when the amino acid sequence encoded by the cDNA is combined with the available amino acid sequence, a complete y2 protein sequence can be obtained. Comparisons, at the nucleotide level, between the known beta- and beta-like globin sequences and the y2 sequence show that the embryonic, fetal-adult duplication occurred approx. 160 million years (MY) ago and that the embryonic-fetal duplication occurred approx. 100 MY ago.     

Nucleotide sequence of cloned cDNA of human apolipoprotein A-I.

ApoA-I is the major human HDL apoprotein. By oligonucleotide hybridization, we have isolated 5 dscDNA clones to human hepatic apo A-I mRNA. One of these clones (pA1-3) was completely sequenced. It has 878 bp plus a poly A tail of 48 and includes all the coding and 3'-untranslated regions of the mRNA and part of the 5'-untranslated region. It predicts a peptide sequence of 267 amino acids (including the 24 amino acid prepropeptides) which is very similar to the sequence reported by Brewer et al., (1978) Biochem. Biophys. Res. Commun. 80:623-630. The predicted signal peptide sequence is highly homologous to the rat apoA-I signal peptide. There is no evidence for any internally repeated segments in apoA-I either at the amino acid or at the DNA level. Using pA1-3 as a probe, we have detected on Northern gels apo A-I mRNA sequences of approximately 1100 nucleotides in human hepatic and baboon hepatic and intestinal RNAs, but not in RNAs from baboon skeletal muscle, kidney or spleen. The demonstration of apo A-I mRNA sequences in specific organs is important to our concept of "reverse cholesterol transport".     

Nucleotide sequence of cloned cDNA specific for rat ribosomal protein L35a

A cDNA clone specific for rat ribosomal protein L35a, which is known to be a tRNA-binding protein, was isolated by hybrid-selected translation from a cDNA library made for 8-9-S poly(A)-rich RNA from regenerating rat liver. The nucleotide sequence of the cDNA was determined. It consists of one base pair from the 5' leading sequence, the entire coding sequence of 333 base pairs and 14 base pairs from the 3' trailing sequence. The primary structure of protein L35a was deduced from the nucleotide sequence. It consists of 109 amino acids with a molecular mass of 12422. The calculated amino acid composition is consistent with that reported for the hydrolysate of L35a. The amino acid sequence showed marked homology with the reported partial sequence of Xenopus leavis ribosomal protein L32, but not significant homology with Escherichia coli ribosomal proteins that bind to tRNA.