Maize chloroplast RNA polymerase: the 78-kilodalton polypeptide is encoded by the plastid rpoC1 gene.

The 180-, 120- and 38-kDa polypeptides found in highly purified maize plastid RNA polymerase preparations are encoded by the maize plastid genes rpoC2, rpoB, and rpoA, respectively [Hu, J. and Bogorad, L. (1990) Proc. Natl. Acad. Sci. USA. 87, pp. 1531-1535]. These genes have segments that specify amino acid sequences homologous to those of E. coli RNA polymerase subunits. The plastid gene products are designated b", b and a, respectively. We report here that the amino-terminal amino acid sequence of a 78-kDa polypeptide also found in highly purified maize plastid RNA polymerase preparations matches precisely the sequence deduced from the maize plastid rpoC1 gene which has segments homologous to the 5' end of the E. coli rpoC gene. Thus, the 78-kDa polypeptide is likely to be a functional component of maize plastid DNA-dependent RNA polymerase. This polypeptide is designated subunit b'. Three polypeptides unrelated to RNA polymerase have also been identified in this preparation.     

The complete amino acid sequence of the A-chain of human plasma alpha 2HS-glycoprotein

Normal human plasma alpha 2HS-glycoprotein has earlier been shown to be comprised of two polypeptide chains. Recently, the amino acid and carbohydrate sequences of the short chain were elucidated (Gejyo, F., Chang, J.-L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R.F., van Halbeck, H., Dorland, L., Gerwig, G. J., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 4966-4971). In the present study, the amino acid sequence of the long chain of this protein, designated A-chain, was determined and found to consist of 282 amino acid residues. Twenty-four amino acid doublets were found; the most abundant of these are Pro-Pro and Ala-Ala which each occur five times. Of particular interest is the presence of three Gly-X-Pro and one Gly-Pro-X sequences that are characteristic of the repeating sequences of collagens. Chou-Fasman evaluation of the secondary structure suggested that the A-chain contains 29% alpha-helix, 24% beta-pleated sheet, and 26% reverse turns and, thus, approximately 80% of the polypeptide chain may display ordered structure. Four glycosylation sites were identified. The two N-glycosidic oligosaccharides were found in the center region (residues 138 and 158), whereas the two O-glycosidic heterosaccharides, both linked to threonine (residues 238 and 252), occur within the carboxyl-terminal region. The N-glycans are linked to Asn residues in beta-turns, while the O-glycans are located in short random segments. Comparison of the sequence of the amino- and carboxyl-terminal 30 residues with protein sequences in a data bank demonstrated that the A-chain is not significantly related to any known proteins. However, the proline-rich carboxyl-terminal region of the A-chain displays some sequence similarity to collagens and the collagen-like domains of complement subcomponent C1q.     

Amino acid sequence of a proline-rich phosphoglycoprotein from parotid secretion of the subhuman primate Macaca fascicularis

The complete amino acid sequence of the macaque proline-rich phosphoglycoprotein (MPRP) was determined by automated Edman degradation of the protein, fragments F-1 and F-2 derived from the protein by an intrinsic salivary protease, and chymotryptic, tryptic, Staphylococcus aureus V8 protease, and endoproteinase lysine-C peptides. MPRP contains 115 amino acid residues including phosphorylated serine at residues 1, 2, 6, 12, and 15, and 6 O-glycosidic carbohydrate units at residues 69, 75, 87 (threonine) and 96, 103, and 106 (serine). The Mr of the polypeptide moiety of the protein is 12,656. The amino-terminal domain contains all 5 phosphoserine residues and most of the other negatively charged and hydrophilic residues, whereas the carboxyl-terminal domain contains 24 of 25 proline residues, and 6 O-glycosidic oligosaccharides. Comparison of MPRP with the four major anionic proline-rich proteins (PRPs) from human glandular secretion shows that 57% of the amino acid residues are identical if gaps are introduced to maximize homology, suggesting that these proteins are phylogenetically related. Significant structural and functional differences occur between the macaque and human proteins. MPRP has 5 phosphoserines, PRPs have 2. MPRP is a glycoprotein, PRPs are not. MPRP inhibits the spontaneous precipitation (primary precipitation) of calcium phosphate salts from supersaturated solutions in addition to inhibiting seeded crystal growth (secondary precipitation) (Oppenheim, F. G., Offner, G. D., and Troxler, R. F. (1982) J. Biol. Chem. 257, 9271-9282), whereas PRPs inhibit only secondary precipitation. MPRP is the only major anionic proline-rich protein in macaque glandular secretion; in contrast, there are four major anionic PRPs and these display a genetic polymorphism. The significance of these structural differences with respect to biological function and the possible relationship of MPRP to salivary mucins are discussed.     

Localization of the gene for human heart fatty acid binding protein to chromosome 1p32-1p33

Among 639 spontaneous abortions between the 8th and 14th week of gestation 342 (53.5%) revealed an abnormal karyotype. While the rate of trisomies distinctly increased with advancing maternal age, a decrease in the rate of 45,X conceptuses and polyploidies was observed among abortions from older women. The overall relation of XXXXXXYY among the tetraploidies was 1411 and that of XXXXXYXYY among the triploidies was 26 361. However, when the latter was related to maternal age, a reversal of the XXXXXY ratio of 12 in the younger to 21 in the older age groups became evident. Furthermore a decrease in the rate of paternally derived partial hydatidiform moles was found among the triploid abortion specimens from older women. From these observations we conclude that digyny plays a major role in the origin of triploidy in the increased maternal age groups, while diandry related to immaturity of oocytes and impairment of oocyte cortical function is more frequent in triploid abortions from younger women.     

Friend strain of spleen focus-forming virus: a recombinant between mouse type C ecotropic viral sequences and sequences related to xenotropic virus.

The genome of the Friend strain of the spleen focus-forming virus (SFFV) has been analyzed by molecular hybridization. SFFV is composed of genetic sequences homologous to Friend type C helper virus (F-MuLV) and SFFV-specific sequences not present in F-MuLV. These SFFV-specific sequences are present in both the Friend and Rauscher strains of murine erythroleukemia virus. The SFFV-specific sequences are partially homologous to three separate strains of mouse xenotropic virus but not to several cloned mouse ecotropic viruses. Thus, the Friend strain of SFFV appears to be a recombinant between a portion of the F-MuLV genome and RNA sequences that are highly related to murine xenotropic viruses. The implications of the acquisition of the xenotropic virus-related sequences are discussed in relation to the leukemogenicity of SFFV, and a model for the pathogenicity of other murine leukemia-inducing viruses is proposed.     

delta-Aminolevulinic acid synthesis in a Cyanidium caldarium mutant unable to make chlorophyll a and phycobiliproteins.

Wild-type cells of the unicellular rhodophyte, Cyanidium caldarium, synthesize chlorophyll a, phycobiliproteins, and heme from δ-aminolevulinic acid during light-dependent chloroplast development but are unable to make photosynthetic pigments in the dark. C. caldarium, mutant GGB-Y, is an obligate heterotroph which, in the light, produces a chloroplast devoid of photosynthetic pigments. The present investigation has shown that δ-aminolevulinic acid is synthesized in cells of mutant GGB-Y incubated with levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase (the second enzyme in the porphyrin biosynthetic pathway). In vivo, cells of mutant GGB-Y preferentially incorporated C₁ of glutamate and α-ketoglutarate into the C₅ fragment (formaldehyde) of δ-aminolevulinic acid after alkaline periodate degradation. This suggested that δ-aminolevulinic acid arises directly from the carbon skeleton of glutamate and α-ketoglutaric acid. The pattern of incorporation of C₃, C₄, and C₅ of α-ketoglutarate into the C₁–C₄ (succinic acid) fragment of δ-aminolevulinic acid after alkaline periodate degradation was consistent with the origin of δ-aminolevulinic acid from a five-carbon precursor. C₁ and C₂ of glycine and C₂ and C₃ of succinate were incorporated into both the formaldehyde and succinate fragments of δ-aminolevulinic acid in a manner inconsistent with condensation of glycine and succinyl CoA by δ-aminolevulinic acid synthetase, the rate-limiting enzyme in the porphyrin pathway in animals and bacteria. Extracts of the soluble protein from cells of mutant GGB-Y displayed a Soret band at 410 nm indicating the presence of hemoproteins. This shows that mutant GGB-Y cells synthesize heme. The respiration of radiolabeled glutamate, α-ketoglutarate, and glycine to ¹⁴CO₂ is consistent with the existence of mitochondrial cytochromes in cells of mutant GGB-Y and with the ability of the mutant to synthesize δ-aminolevulinic acid. The present results suggest that δ-aminolevulinic acid is synthesized directly from glutamate or α-ketoglutarate and that this is the only process by which the rate-limiting intermediate in the porphyrin pathway is synthesized in C. caldarium. If correct, the rate-limiting, regulative enzyme in the biosynthetic pathway for synthesis of chlorophyll a, bile pigment (phycocyanobilin), and heme must have been completely different in the evolutionary antecedents of modern-day plants and animals.     

Spleen focus-forming Friend virus: identification of genomic RNA and its relationship to helper virus RNA.

The genome of the defective, murine spleen focus-forming Friend virus (SFFV) was identified as a 50S RNA complex consisting of 32S RNA monomers. Electrophoretic mobility and the molecular weights of unique RNase T1-resistant oligonucleotides (T1-oligonucleotides) indicated that the 32S RNA had a complexity of about 7.4 kilobases. Hybridization with DNA complementary to Friend murine leukemia virus (Fr-MLV) has distinguished two sets of nucleotide sequences in 32S SFFV RNA, 74% which were Fr-MLV related and 26% which were SFFV specific. By the same method, SFFV RNA was 48% related to Moloney MLV. We have resolved 23 large T1-oligonucleotides of SFFV RNA and 43 of Fr-MLV RNA. On the basis of the relationship between SFFV and Fr-MLV RNAs, the 23 SFFV oligonucleotides fell into four classes: (i) seven which had homologous equivalents in Fr-MLV RNA; (ii) six more which could be isolated from SFFV RNA-Fr-MLV cDNA hybrids treated with RNases A and T1; (iii) eight more which were isolated from hybrids treated with RNases A and T1; and (iv) two which did not have Fr-MLV-related counterparts. Surprisingly, the two class iv oligonucleotides had homologous counterparts in the RNA of six amphotropic MLV's including mink cell focus-forming and HIX-MLVs analyzed previously. The map locations of the 23 SFFV T1-oligonucleotides relative to the 3' polyadenylic acid coordinate of SFFV RNA were deduced from the size of the smallest polyadenylic acid-tagged RNA fragment from which a given oligonucleotide was isolated. The resulting oligonucleotide map could be divided roughly into three segments: two terminal segments which are mosaics of oligonucleotides of classes i, ii, and iii and an internal segment between 2 and 2.5 kilobases from the 3' end containing the two oligonucleotides shared with amphotropic MLVs. Since SFFV RNA consists predominantly of sequence elements related to ecotropic and amphotropic helper-independent MLVs, it would appear that the transforming gene of SFFV is not a major specific sequence unrelated to genes of helper viruses, as is the case with Rous sarcoma and probably withe other defective sarcoma and acute leukemia viruses.     

Characterization of a protein found in cells infected with the spleen focus-forming virus that shares immunological cross-reactivity with the gp70 found in mink cell focus-inducing virus particles.

Previously we detected an antigen in cells infected with the spleen focus-forming virus (SFFV) with a radioimmunoassay specific for the gp 70's of murine leukemia mink cell focus-inducing (MCF) viruses. This antigen has now been characterized in competition radioimmunoassays with limiting dilutions of antibody and in pulse-labeling studies under conditions of antibody excess. Both methods of analysis indicate that the SFFV-encoded antigen is a glycoprotein with a molecular weight of approximately 52,000. The gp52 shared immunological reactivity and methionine-containing tryptic peptides with the gp70 of a Friend MCF virus and was expressed on the surface of SFFV-infected cells as well as in the cytoplasm. The gp52 could be detected (i) in fibroblastic cell lines from several species when these cells were infected with SFFV; (ii) in several established erythroleukemic cell lines; and (iii) in the spleens of mice recently infected with SFFV. Although it shared immunochemical properties with the gp70 of Friend MCF virus, the gp52 could be distinguished from the MCF gp70 (i) by its apparent lack of group and interspecies immunological determinants compared with MCF virus-derived gp70's; (ii) by its failure to be released from cells infected with SFFV or SFFV plus helper virus; (iii) by its molecular weight; and (iv) by tryptic peptide analysis. The results indicate that SFFV codes for an MCF gp70-related gp52 which is apparently no longer a virion structural protein like the MCF gp70 from which it was originally derived.     

Bile pigment synthesis in plants. Incorporation of haem into phycocyanobilin and phycobiliproteins in Cyanidium caldarium.

A procedure was developed whereby haem was taken up by dark-grown cells of the unicellular rhodophyte Cyanidium caldarium. These cells were subsequently incubated either in the dark with 5-aminolaevulinate, which results in excretion of phycocyanobilin into the suspending medium or incubated in the light, which results in synthesis and accumulation of phycocyanin and chlorophyll a within the cells. Phycocyanobilin was isolated from phycocyanin by cleavage from apoprotein in methanol. Phycocyanobilin prepared from phycocyanin or excreted from cells given 5-aminolaevulinate was methylated and purified by t.l.c. By using 14C labelling either in the haem or in 5-aminolaevulinate administered, haem incorporation into phycocyanobilin was demonstrated in both dark and light systems. Since chlorophyll a synthesized in the light in the presence of labelled haem contained no radioactivity, it was clear that haem was directly incorporated into phycocyanobilin and not first converted into protoporphyrin IX. These results clearly demonstrate phycocyanobilin synthesis via haem and not via magnesium protoporphyrin IX as has also been postulated.