Alan Przybyla

Messenger RNA for myosin polypeptides: Isolation from single myogenic cell cultures

Messenger RNA which stimilates the synthesis of myosin heavy chain in a reticulocyte lysate has been isolated from single myogenic cell cultures. Specific myosin polypeptides have been identified by immunoprecipitation with an antibody made to purified adult chicken skeletal muscle myosin. This mRNA binds to oligo(dT)-cellulose, and an active fraction from sucrose gradients migrates as 26S on formamide-polyacrylamide gels. The relative amount of this RNA increases dramatically at the time of terminal differentiation.

Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase

Formyltetrahydrofolate synthetase (FTHFS) (EC 6.3.4.3), a thermostable protein of four identical subunits from Clostridium thermoaceticum was cloned into Escherichia coli SK1592. The clone (CRL47) contained a 9.5 kb EcoRI fragment of C. thermoaceticum DNA ligated into pBR322. It produced catalytically active, thermostable FTHFS, that was not found in E. coli SK1592 containing native pBR322. The identity of the expressed enzyme was confirmed by specific binding of rabbit polyclonal anti-FTHFS serum produced against C. thermoaceticum FTHFS. The specific activities (μmol·min-1·mg-1) of FTHFS in cell free extracts of CRL47 were 28–89 when assayed at 50°C and pH8. This was from 3–10-fold higher than in C. thermoaceticum extracts. FTHFS was purified to homogeneity from CRL47. The purified enzyme behaved during electrophoresis and gel chromatography and it had similar specific activity and thermostability as the enzyme purified from C. thermoaceticum.

Carboxy-terminal processing of the large subunit of [NiFe] hydrogenases

Two electrophoretic forms of the large subunit of the soluble periplasmic [NiFe] hydrogenase from Desulfovibrio gigas have been detected by Western analysis. The faster moving form co‐migrates with the large subunit from purified, active enzyme. Amino acid sequence and composition of the C‐terminal tryptic peptide of the large subunit from purified hydrogenase revealed that it is 15 amino acids shorter than that predicted by the nucleotide sequence. Processing of the nascent large subunit occurs by C‐terminal cleavage between His536 and Val537, residues which are highly conserved among [NiFe] hydrogenases. Mutagenesis of the analogous residues, His582 and Val583, in the E. coli hydrogenase‐1 (HYD1) large subunit resulted in significant decrease in processing and HYD1 activity.

Differential expression of aortic and lung elastin genes during chick embryogenesis.

Abstract The ratios of tropoelastin b to a were measured in chick aorta and lung during embryogenesis. The rates of tropoelastin a and b synthesis were determined in short-term organ culture. The results demonstrated that in lung tissue the ratio of the two tropoelastins remained essentially constant. Each of the tropoelastins comprised 50% of the total elastin synthesis. In the aortic tissue, tropoelastin b represented 70% of the total elastin in the 11- to 13-day embryos and increased to 91% by Day 16. These observations seen in the organ culture system were paralleled in measurements of functional mRNAs coding for the two proteins. Measurements of functional tropoelastin mRNAs from both lung and aortic tissues were performed in a mRNA-dependent rabbit reticulocyte lysate system. Although the changes in the abundance of the tropoelastin mRNAs revealed the same trend as that seen in the organ culture data, the magnitude of the tropoelastin b to a ratio in the aortic organ culture was twice that determined in the cell-free translation of aortic mRNAs. The data obtained from both cell-free translations and organ culture experiments demonstrate that there is a differential expression of elastin genes during aorta development which is significantly different from that found in developing lung.

IDENTIFICATION OF THREE CLASSES OF HYDROGENASE IN THE GENUS, DESULFOVIBRIO

A comparison of amino-terminal amino acid sequences from the large and small subunits of hydrogenases from Desulfovibrio reveals significant differences. These results, in conjunction with antibody analyses, clearly indicate that the iron, iron + nickel, and iron + nickel + selenium containing hydrogenases represent three distinct classes of hydrogenase in Desulfovibrio.

Cloning, sequencing and overexpression of the Desulfovibrio gigas ferredoxin gene in E. coli

We have cloned the gene encoding Desulfovibrio gigas ferredoxin using a photodigoxigenin‐labelled probe synthesized with the polymerase chain reaction. The DNA sequence of the gene predicts a polypeptide of 58 residues after removal of the initial formyl methionine (polypeptide M r = 6,276). The ferredoxin gene was expressed in aerobically grown E. coli behind the lac promoter of pUC18 resulting in a high level of ferredoxin expression which comprises about 10% of the total cell protein. EPR analysis of recombinant ferredoxin revealed the presence of a [3Fe‐4S] cluster which is characteristic of native D. gigas ferredoxin II.

Phosphorylation pattern of a 25 Kdalton stress protein from rat myoblasts

Phosphorylation of a 25 Kdalton nuclear stress protein from rat muscle was examined by two-dimensional gel electrophoresis and one-dimensional peptide mapping. These studies show that three 25 Kdalton stress proteins found in stressed rat myoblasts are actually the same protein with charge variation brought about by multiple phosphorylations. Furthermore, the predominant charge variant of 25 Kdalton protein found in cells is dependent on the intensity of the stress applied to cells.

Optimal conditions for cell-free synthesis of elastin

Optimal conditions for the translation of elastin mRNA1 in a mRNA-dependent rabbit reticulocyte lysate were determined. Using total RNA isolated from embryonic chick aortae as the source of exogenous RNA, the concentrations of various components present in the translation assay were varied and the effect on elastin synthesis quantitated by immunoprecipitation. Components examined included: magnesium acetate, potassium chloride, spermidine, creatine phosphate, ATP, and GTP. In addition, it was found that heating of the RNA prior to translation significantly enhanced total protein synthesis, elastin synthesis, and the synthesis of proteins possessing molecular weights of greater than 80,000.

[41] Cell-free translation of elastin mRNAs

Publisher Summary To cover fully the system for effective cell-free translations of elastin mRNAs, the methodology is divided into four sections dealing with (1) isolation of RNA and preparation of mRNA, (2) preparation of the mRNA-dependent reticulocyte lysate and optimal conditions for the cell-free translation of elastin mRNAs, (3) identification of tropoelastins in a cell-free translation assay, and (4) quantitation of tropoelastins in the cell-free translation system. It is important to note that the discussion of the cell-free synthesis of elastin involves the synthesis of two proteins designated tropoelastins a and b. All the evidence to date suggests strongly that these two tropoelastins are separate gene products. Consequently, the methodologies described are geared not only for the production of soluble elastin, but also for the separate identification of tropoelastins a and b.