Nucleotide sequence and derived amino acid sequence of a cDNA encoding human muscle carbonic anhydrase

We report the nucleotide (nt) sequence of a full length cDNA clone, pCA15, which encodes the human muscle-specific carbonic anhydrase, CAIII. pCA15 identifies a 1.7-kb mRNA, which is present at high levels in skeletal muscle, at much lower levels in cardiac and smooth muscle and which appears to be developmentally regulated. The CAIII mRNA is distinguished by a 887-nt long 3'-untranslated region, containing two AAUAAA signal sequences and is longer than either of the mRNAs encoding the erythrocyte CAs, CAI and CAII, which each have relatively shorter 3'-untranslated regions, 360 and 670 nt long, respectively. The derived amino acid (aa) sequence for human CAIII shows 85% homology with ox CAIII, 62% homology with human CAII and 54% with human CAI when simple pairwise aa comparisons are made. We describe an allelic variation at a TaqI restriction site for CAIII which occurs at high frequency in the European population.     

Nucleotide and derived amino acid sequence of the subtilisin from the antarctic psychrotroph Bacillus TA39.

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph Bacillus TA39 was determined. The primary structure of the subtilisin precursor is composed of 420 amino acids giving rise to a mature enzyme of 309 amino acids. Asp-145, His-185 and Ser-361 are the proposed catalytic residues of the active site.     

N-terminal amino acid sequence of leukemia derived growth factor (LGF) from human erythroleukemia cell culture

Summary A human erythroleukemia cell line, K-562 T1, was adapted to a protein-free chemically defined medium (1); that is, the medium does not contain any proteins such as exogenous hormones, growth factors, serum and serum albumin. The K-562 T1 cells which can proliferate in a protein-free medium are one of the model systems suitably supporting the autocrine hypothesis (2), which claims that cancer cells produce and respond to their own growth factors (3). The K-562 T1 cells were cultured in a protein-free medium at large scale and the growth factors were purified from the conditioned medium. It was found that K-562 T1 cells produce at least two growth factors; one is LGF-I (leukemia-derived growth factor-I) which can stimulate the proliferation of a wide range of human leukemia cell lines and the other is LGF-II (leukemia-derived growth factor-II), which can contribute to the growth of fibroblasts. LGF-I was purified using QAE-Sephadex, Bio Gel P-60 and Mono S FPLC. The purified protein was found to be homogenous by SDS-polyacrylamide gel electrophoresis and NH₂-terminal sequence analysis. The molecular weight of LGF-I was 20,000 by SDS-polyacrylamide gel electrophoresis. The 30 NH₂-terminal residues of LGF-I are the same as that of ubiquitin. Ubiquitin is a protein found in eukaryotic cells with molecular weight of 8,600. In the nucleus ubiquitin is conjugated to histone 2A to form the nuclear protein A24 which may play a role in regulation of chromatin structure (3), and in the cytoplasm is part of an ATP-dependent non-lysosomal proteolytic pathway (4). However, its physiological significance has not yet been fully resolved. Ubiquitin purified from bovine thymus did not show cell proliferating activity for any cells tested. The results suggest that LGF-I is a new autocrine growth factor with a molecular weight of 20,000 daltons, containing ubiquitin at the NH₂-terminal end. This work was supported by funds obtained under the Research and Development Project of Basic Technologies for Future Industries from the Ministry of International Trade and Industry of Japan. Editor’s Statement Identification of sequences identical to ubiquitin associated with the leukemia-derived growth factor described in this report is particularly intriguing considering recent reports of association of ubiquitin with surface membrane receptors of lymphocytes and fibroblasts. References: SCIENCE vol. 231, pgs, 823–829; NATURE vol. 323, pgs 226–232, 1986. David W. Barnes     

Nucleotide sequence of complementary DNA and derived amino acid sequence of murine complement protein C3

The nucleotide sequences coding for murine complement component C3 have been determined from a cloned genomic DNA fragment and several overlapping cloned complementary DNA fragments. The amino acid sequence of the protein was deduced. The mature beta and alpha subunits contain 642 and 993 amino acids respectively. Including a 24 amino acid signal peptide and four arginines in the beta-alpha transition region, which are probably not contained in the mature protein, the unglycosylated single chain precursor protein preproC3 would have a molecular mass of 186 484 Da and consist of 1663 amino acid residues. The C3 messenger RNA would be composed of a 56 +/- 2 nucleotide long 5' non-translated region, 4992 nucleotides of coding sequence, and a 3' non-translated region of 39 nucleotides, excluding the poly A tail. The beta chain contains only three cysteine residues, the alpha chain 24, ten of which are clustered in the carboxy terminal stretch of 175 amino acids. Two potential carbohydrate attachment sites are predicted for the alpha chain, none for the beta chain. From a comparison with human C3 cDNA sequence (of which over 80% has been determined) an extensive overall sequence homology was observed. Human and murine preproC3 would be of very similar length and share several noteworthy properties: the same order of the subunits in the precursor, the same basic residue multiplet in the beta-alpha transition region, and a glutamine residue in the thioester region. The equivalent position of the known factor I cleavage sites in human C3 alpha could be located in the murine C3 alpha chain and the size and sequence of the resulting peptide were deduced. A comparison of the amino acid sequences of murine C3 and human alpha 2-macroglobulin is given. Several areas of strong sequence homology are observed, and we conclude that the two genes must have evolved from a common ancestor.     

Nucleotide and primary amino acid sequence of porcine lactoferrin.

A cDNA encoding porcine lactoferrin (pLF) was isolated from a porcine mammary gland lambda gt11 cDNA library using human lactoferrin cDNA as the hybridization probe. Nucleotide sequence analysis indicates that pLF is 686 amino acids in length and shares 72.6%, 70.7% and 62.2% overall amino acid sequence identity with bovine, human and murine lactoferrin, respectively.     

Purification to homogeneity and NH2-terminal amino acid sequence of a novel interleukin 1 species derived from a human B cell line

A subclone, referred to as 3B6, derived from a DR-negative EBV-transformed B cell line, has been found to spontaneously produce IL 1. 3B6-IL 1 displays a pI of 5 on FPLC chromatofocusing. It has been purified to homogeneity by a sequence of ion-exchange chromatography and affinity chromatography on procion red agarose. The homogeneous material migrated with an apparent m.w. of 13,500 on SDS-PAGE. The overall recovery of IL 1 activity was estimated at 57%. The final material had a specific activity of 7.8 X 10(6) half-maximal units/mg and represented a 50,000-fold purification. A partial NH2-terminal amino acid sequence has been obtained that is different from those reported from monocytic IL 1. However, this molecule can formally be identified as IL 1 on its spectrum of biologic activities. In addition to inducing the proliferation of murine thymocytes in the co-stimulator assay. 3B6-IL 1 is active on both human T and B cells, respectively, in inducing IL 2 synthesis by cells from a subcloned HSB 2 line and promoting the proliferation of anti-IgM-stimulated human peripheral blood B lymphocytes. Furthermore, 3B6-IL 1 acts as a growth factor for normal human fibroblasts and for the 3B6 line itself. However, 3B6-IL 1 is not pyrogenic in rabbits. Thus, the 3B6 cell line was shown to produce a new molecular species of IL 1, with respect to its NH2-terminal sequence, which shared all of the studied biologic activities of monocytic IL 1 except for pyrogenicity.     

Nucleotide sequence of the bacteriophage T4 gene 57 and a deduced amino acid sequence.

A 693 basepair cloned fragment of bacteriophage T4 DNA, which supports specifically growth of T4 amber mutants in gene 57, has been sequenced. A polypeptide can be deduced from this sequence, that is either 54 or 60 amino acids long depending which of two AUG codons, 18 nucleotides apart, are used for initiation. The size of this deduced polypeptide is compatible with the size of a single polypeptide (based on polyacrylamide gel electrophoresis) synthesized in vivo in E. coli under the direction of the cloned T4 DNA fragment.     

The amino acid sequence of a hypothalamic peptide, neurotensin.

The amino acid sequence of neurotensin, a hypotensive peptide isolated from acid-acetone extracts of bovine hypothalami, has been established as less than Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-Oh. (The nomenclature and symbols follow the suggestions of the IUPAC-IUB Commission on Biochemical Nomenclature (1972) J. Biol. Chem. 247, 977). This was accomplished by sequence analyses performed on the intact peptide as well as its isolated tryptic, chymotryptic, and papain-generated fragments. The results of enzymic hydrolyses were consistent with the specificities of the enzymes used and indicated that all of the amino acids are unsubstituted and in the L configuration. The absence of non-amino acid constituents was further supported by analyses of electrophoretic mobility-molecular weight relationships of neurotensin and its fragments.