Structure of murine complement component C3. I. Nucleotide sequence of cloned complementary and genomic DNA coding for the beta chain

The nucleotide sequence coding for the beta chain of murine C3 was determined from cloned cDNA and genomic DNA fragments. Sonicated subfragments were randomly inserted into the bacteriophage M13 and sequenced using the dideoxynucleotide technique. Each nucleotide was sequenced on average six times in these studies. The derived amino acid sequence includes a signal peptide and a tetra-arginine sequence between the beta and alpha subunits in the precursor polypeptide prepro-C3. Together with the accompanying report (Wetsel, R.A., Lundwall, A., Davidson, F., Gibson, T., Tack, B.F., and Fey, G.H. (1984) J. Biol. Chem. 259, 13857-13862), this paper completes the analysis of the coding sequences for the prepro-C3 polypeptide. The derived molecular weight of the unglycosylated beta chain (642 amino acids) is 70,641. The sequences of the first two introns in the murine C3 gene and of the 5'-flanking 106 nucleotides are also reported. The 5'-flanking region contains a TATA consensus sequence in agreement with an earlier report (Wiebauer, K., Domdey, H., Diggelmann, H., and Fey, G.H. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7077-7081), presumed to be involving in regulating the expression of the C3 gene. A striking feature of the derived sequence was that only 3 cysteine residues were found, all located in the C-terminal part of the polypeptide chain. No carbohydrate attachment sites were predicted in the beta chain.     

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 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 cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones

Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.     

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.     

Sequence heterogeneity of murine complementary DNA clones related to the C4 and C4-Slp isoforms of the fourth complement component

Two classes of mRNA encoding the murine C4 protein were identified by sequence analysis of clones isolated from a liver complementary DNA library. The divergence found within a 357 base pair sequence available for comparison is limited to five nucleotide replacements located in the region corresponding to the carboxy-terminal end of the C4d peptide fragment. One of the nucleotide substitutions influences the presence of a site for the Hind III restriction endonuclease. That this restriction site indeed discriminates the two non-allelic genes encoding the mouse C4 and C4-Slp isoforms has been demonstrated by Southern blot analysis and nucleotide sequencing at the genomic level. Circumstantial evidence supports the identification of the gene lacking the Hind III site in the region corresponding to the carboxy-terminal end of the C4d fragment as the one encoding the C4-Slp isotype.     

Nucleotide sequence of cloned cDNA coding for pumpkin 11-S globulin beta subunit

cDNA coding for preproglobulin beta, a precursor protein of 11-S globulin beta subunit, was cloned and the nucleotide sequence has been determined. The sequence covers the whole coding region (1440 base pairs) with 5' and 3' noncoding region (30 and 214 base pairs, respectively). The deduced amino acid sequence of preproglobulin beta consists of a 21-amino-acid N-terminal signal peptide, preceding the acidic gamma polypeptide region (275 amino acids) and the subsequent basic delta region (184 amino acids). The site for post-translational cleavage of the precursor polypeptide to make the gamma and delta chains is estimated to be located between the asparagine-glycine residues. The N-terminal amino acid of the gamma chain of mature 11-S globulin beta subunit was reported to be blocked by 5-oxoproline (pyroglutamic acid) [Ohmiya et al. (1980) Plant Cell Physiol. 21, 157-167]. It was shown that the blocked N-terminal amino acid is coded as a glutamine residue. The derived amino acid sequence was also compared with those of precursor proteins of other 11-S globulins such as soybean glycinin, cotton beta globulin, pea legumin and rape 11-S globulin by dot matrix analysis.     

Structure of the Drosophila DNA topoisomerase II gene. Nucleotide sequence and homology among topoisomerases II

We have determined the nucleotide sequence of the Drosophila DNA topoisomerase II gene. Data from primer extension and S1 nuclease protection experiments were combined with comparisons of genomic and cDNA sequences to determine the structure of the mature messenger RNA. This message has a large open reading frame of 4341 nucleotides. The length of the predicted protein is 1447 amino acids with a molecular weight of 164,424. Topoisomerase II can be divided into three domains: (1) an N-terminal region with homology to the B (ATPase) subunit of the bacterial type II topoisomerase, DNA gyrase; (2) a central region with homology to the A (breaking and rejoining) subunit of DNA gyrase; (3) a C-terminal region characterized by alternating stretches of positively and negatively charged amino acids. DNA topoisomerase II from the fruit fly shares significant sequence homology with those from divergent sources, including bacteria, bacteriophage T4 and yeasts. The location and distribution of homologous stretches in these sequences are analyzed.     

Nucleotide sequence involved in the replication of cloned yeast mitochondrial DNA

A fragment of yeast mitochondrial DNA, Alu B, has two subfragments, Alu B1 and Alu B2. They were each cloned and sequenced. The autonomously replicating function of the curtailed Alu B1 (342 bp) was defined within 186 bp. A GC-rich sequence identical to the oris sequence in the curtailed Alu B1 was unnecessary for its autonomously replicating function. The 186 bp sequence had an ATATAAAT sequence and the stem and loop structures. The base sequence of Alu B2 also contained the same octanucleotides, the stem and loop structures, one oris sequence and one unique GC cluster. Yeast transformants with cloned Alu B2 grew slowly. The cloned Alu B2 was enlarged in the yeast host concomitantly with compensation of the slow growth of the transformants.     

Nucleotide sequence of a highly repetitive component of rat DNA.

A highly repetitive component of rat DNA which could not yet be enriched by density gradient centrifugation was isolated with the help of the restriction nuclease Sau3AI. This nuclease converted the bulk of the DNA to small fragments and left a repetitive DNA component as large fragments which were subsequently purified by gel filtration and electrophoresis. This DNA component which was termed rat satellite DNA I is composed of tandemly repeated 370 bp blocks. According to sequence analysis the 370 bp repeats consist of alternating 92 and 93 bp units with homologous but not identical sequences. Methylation of CpG residues was correlated to the rate of cleavage by restriction nucleases. Significant homologies exist between the sequences of rat satellite DNA I and satellite DNAs of several other organisms. The divergence of the sequence of rat satellite DNA I was discussed with respect to evolutionary considerations.