Biomedical subjects

R C Sowder

Publications and source records attributed to R C Sowder.

40 records · Page 3Linked to original sources

PubMed1987

Characterization of mouse mammary tumor virus gag-pro gene products and the ribosomal frameshift site by protein sequencing.

The synthesis of retroviral polyproteins that are the translational products of the genome-size mRNA is initiated in the upstream gag gene. The synthesis of the products of the protease gene (pro) and polymerase gene (pol) is regulated by translational suppression (in-frame read-through or frameshift) of termination codons as a strategy developed for controlling the level of replicative enzymes required only in catalytic amounts. In mouse mammary tumor virus (MMTV), three overlapping reading frames are utilized for the synthesis of gag-encoded Pr77, gag-pro-encoded Pr110, and gag-pro-pol-encoded Pr160 polyproteins. To characterize pro gene products and to determine the site of frameshift required for the synthesis of Pr110, we purified and sequenced three MMTV proteins: p14, p30, and p13. Sequence analysis showed that p14 is the basic nucleic acid-binding protein derived entirely from gag, and p13 is a product of the pro gene and has characteristic sequences of protease. A comparison of the amino acid sequences of p30 with the corresponding nucleotide sequence of proviral DNA allowed the delineation of the frameshift site utilized in vivo for the synthesis of the gag-pro-encoded fusion polyprotein Pr110. The results showed that: (i) the N-terminal 94 residues of p30 are translated from the gag frame; (ii) residue leucine-95 is specified by either the last UUG codon of gag or the overlapping CUU codon in the pro frame; and (iii) the elongation of the peptide chain from residue 96 continued to be encoded in the pro frame to the pro terminator. The possible mechanisms of frameshift and of the tRNAs involved are discussed.

Amino Acid Sequence↗

PubMed1987

Chemical and immunological characterizations of equine infectious anemia virus gag-encoded proteins.

The viral core proteins (p15, p26, p11, and p9) of equine infectious anemia virus (EIAV) (Wyoming strain) were purified by reverse-phase high-pressure liquid chromatography. Each purified protein was analyzed for amino acid content, N-terminal amino acid sequence, C-terminal amino acid sequence, and phosphoamino acid content. The results of N- and C-terminal amino acid sequence analysis of each gag protein, taken together with the nucleotide sequence of the EIAV gag gene (R. M. Stephens, J. W. Casey, and N. R. Rice, Science 231:589-594, 1986), show that the order of the proteins in the precursor is p15-p26-*-p11-p9, where a pentapeptide also found in the virus is represented by the asterisk. The data are in complete agreement with the predicted structure of the gag polyprotein and show the peptide bonds cleaved during proteolytic processing. The N-terminus of p15 is blocked to Edman degradation. The p11 protein is identical to the nucleic acid-binding protein of EIAV previously isolated (C. W. Long, L. E. Henderson, and S. Oroszlan, Virology 104:491-496, 1980). High-titer rabbit antiserum was prepared against each purified protein. These antisera were used to detect the putative gag precursor (Pr55gag) and intermediate cleavage products designated Pr49 (p15-p26-*-p11), Pr40 (p15-p26), and Pr35 (p26-*-p11) in the virus and in virus-infected cells. High-titer antisera to EIAV p15 and p26 showed cross-reactivity with the homologous protein of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus.

Amino Acid Sequence↗

PubMed1983

A rapid, large scale purification procedure for gibbon interleukin 2.

Interleukin 2 (IL 2) was purified from the conditioned medium of a gibbon T cell line, MLA144, which releases IL 2 constitutively. The IL 2 was obtained free of contaminating proteins by a simple process consisting of an initial batch purification on trimethylsilyl-controlled pore glass followed by reversed phase high pressure liquid chromatography. Overall recovery of IL 2 activity ranged from 70 to 100% of initial activity and yielded 2 X 10(6) or greater units of IL 2 per 15 liters of serum-free MLA144 conditioned medium. The specific activity of purified IL 2 ranged from 0.5 to 1 X 10(8) IL 2 U/mg protein. The purified IL 2 showed four molecular species when analyzed by two-dimensional isoelectric focusing-SDS-polyacrylamide gel electrophoresis. Each of the four molecular forms was active in the bioassay for IL 2 activity. Three molecular forms had apparent m.w. of 16,000 but different isoelectric points of 5.9, 6.3, and 6.7. One molecular form had an apparent m.w. of 15,000 and an isoelectric point of 7.2. The most abundant form of IL 2 had an apparent m.w. of 16,000 and an isoelectric point of 6.3. The purified IL 2 supported the growth of IL 2-dependent lymphocytes to a greater extent than did the same level of crude IL 2-containing MLA144 conditioned medium. The ability to purify large amounts of IL 2 by a rapid and efficient procedure will be of great help in both biochemical and immunologic studies of this lymphokine.

Amino Acids↗

PubMed1981

Primary structure of the low molecular weight nucleic acid-binding proteins of murine leukemia viruses.

Murine leukemia viruses contain a low molecular weight basic protein, designated p10, which binds to single-stranded nucleic acids. The complete amino acid sequence of p10 from the Rauscher strain of virus has been determined. The partial amino acid sequences of p10s from Moloney, Friend, AKR, Gross, radiation leukemia, and BALB/2 viral strains have also been determined using microsequencing techniques. Rauscher p10 is composed of 56 amino acid residues; the other p10s are similar in size but differ from Rauscher by a few conservative amino acid substitutions. The structure of Rauscher p10 was compared to the structure of a functionally homologous protein from Rous avian sarcoma virus. The comparison revealed regions of amino acid sequence homologies which indicate a phylogenetic relationship between the murine and avian viral strains. The analyses revealed a periodic placement of three Cys residues and a Gly-His sequence. A structure involving these residues is found once in the murine protein and twice in the avian protein. A similar structure is seen in the single stranded nucleic acid binding protein of bacteriophage T4. However, in the latter case, the order of amino acid residues is inverted.

AKR murine leukemia virus↗