rII cistrons of bacteriophage T4. DNA sequence around the intercistronic divide and positions of genetic landmarks

An 873 base-pair DNA sequence from the rII region of bacteriophage T4 is presented. The sequence encodes 139 carboxyl-terminal amino acids of rIIA and the amino-terminal 146 amino acids of rIIB. Eleven base-pairs separate the rIIA stop codon (UAA) and the rIIB AUG.An extensive genetic map is superimposed on the DNA sequence, showing the deduced locations of many of the mutations (base-pair substitutions, frameshifts, deletions) found in previous rII genetic studies.     

Changes in the abundance of polyadenylated RNA during slime mould development measured using cloned molecular hybridization probes.

Total Dictyostelium discoideum messenger RNA prepared from cells at the eighth hour of development in suspension culture has been copied into DNA. This DNA was inserted into the plasmid PMB9 and used to transform Escherichia coli. The resulting “clone bank” was screened using an in situ hybridization technique in which replicate copies of a set of clones were hybridized with mRNA isolated from vegetative (non-developing) cells and from cells at the eighth hour of development. The mRNA was labelled in vitro so that the amount of hybridization to a given clone is a measure of the relative abundance of the mRNA complementary to the DNA in that clone. By comparing the amount of hybridization of the mRNA preparations to each clone, it has been possible to identify plasmids containing D. discoideum DNA whose complementary mRNA increases or decreases in abundance during development. These observations are direct proof of a change in mRNA concentration during D. discoideum development for individual high and medium abundance mRNA species. We can estimate from these results the proportion of such mRNA species whose concentration increases significantly during development and we find that only a small fraction show such a change.     

Lipid phase transitions in model biomembranes: The effect of ions on phosphatidylcholine bilayers

Differential scanning calorimetry has been used to study the endothermic phase behaviour of some model biomembranes (i.e. phosphatidylcholine-water systems) in the presence of a wide range of alkaline, alkaline earth and heavy metal salts. Studies and comparisons were made of both cation and anion effects. Shifts occur in the temperatures of both the pre-transition and main transition endotherms. The observed shifts are smaller than those which have been reported for charged lipids, and no evidence has been found for the formation of specific complexes. Electron microscopic studies on freeze-fractured dispersions of phosphatidylcholine-water-salt systems show that with some salts the typical rippled surface observed with l-α-dimyristoyl phosphatidylcholine, when in the gel state, is replaced by a smooth surface.     

Lactate dehydrogenase-C mRNA: Its isolation and invitro translation

Lactate dehydrogenase-C (LDH-C) mRNA was purified from $\text{DBA}\text{2}$ mouse testes and translated $\text{in}\text{vitro}$. First, the LDH-C synthesizing polysomes were isolated by double immunoprecipitation using specific anti-LDH-C and anti-horse immunoglobulin antibodies. Extraction of mRNA was made from the isolated polysomes using hot sodium dodecyl sulfate-phenol method at alkaline pH. In a wheat germ cell-free translation system, the mRNA coded for a polypeptide chain that could be immunoprecipitated with specific anti LDH-C antibody and comigrated with authentic LDH-C in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Enzymatic repair of O-alkylated thymidine residues in DNA: involvement of a O4-methylthymine-DNA methyltransferase and a O2-methylthymine DNA glycosylase

The distribution and intracellular translocation of AFB1 in various subcellular fractions was investigated in isolated hepatocytes by pulse-chase experiments. After labeling the hepatocytes with [3H]-AFB1 (14.5 nM) for 15 min, the highest concentration of [3H]-AFB1 was found in the cytosolic fraction where 66% was bound noncovalently and 1.5% covalently. The lowest concentration of [3H]-AFB1 was found in the nuclear fraction; 36% and 4.9% were bound noncovalently and covalently respectively. When the [3H]-AFB1 loaded cells were chased with unlabeled AFB1 (1 microM), the radioactivity of [3H]-AFB1 in the cell lysate and cytosolic fraction decreased in time with an apparent rate of elimination (t1/2) of 93 min and 66 min, respectively. The levels of covalently bound AFB1 increased with time and reached a maximum at 60 min in nuclei (270%), and at 120 min in mitochondria (220%) and cytosol (430%) as compared to the zero time. Only in the microsomal fraction was there no significant increase with time in covalently bound AFB1. These results suggest that the toxin after activation by the microsomal mixed function oxidases was either detoxified or transported to other cellular organelles where covalent binding of macromolecules occurred.     

Arrangement of the single-stranded fragments in E. coli bacteriophage BF23 DNA

Bacteriophage BF23st(0) DNA was denatured with alkali and fractionated by agarose gel electrophoresis. Seven single-stranded fragments (designated Fragments I--VII) were identified as the major constituents of the phage DNA. The presence of several minor fragments which represent minor populations of the phage genome was also observed. The largest fragment (Fragment I) represents the intact strand of phage DNA, whereas the other fragments form the complementary strand. Thus, BF23st(0) DNA carries single-strand interruptions in only one strand. The arrangement of the major fragments in the nicked strand was determined by use of gamma-exonuclease and agarose gel electrophoresis. From the mode of action of this nuclease, and from the kinetics of release or disappearance of the fragments, the polarity of the fragments in BF23st(0) DNA was specified. In addition, the presence of two types of major phage populations differing in their composition of the fragments was demonstrated. One type has an additional nick (yielding Fragment IV and Fragment V) in a specific fragment (Fragment II) of other type.     

Unscheduled DNA synthesis induced in mouse spermatids after combined treatment with methyl methanesulfonate and X-rays.

Unscheduled DNA synthesis (UDS), which is considered to be DNA repair, has been studied in early- to mid-spermatid stages of the mouse after combined treatments with X-rays and methyl methanesulfonate (MMS). UDS in spermatids was detected by giving testicular injections of [methyl-3H]thymidine ([3H]dThd) and making use of the fact that no scheduled DNA synthesis occurs in the germ cells after the last S period in primary spermatocytes. X-rays and MMS are each able to induce UDS in mouse spermatids. However, there was a statistically significant reduction in the amount of UDS observed when X-ray exposures of from 200 to 600 R were given 4 h before an i.p. injection of 75 mg/kg of MMS and concurrent testicular injections of [3H]dThd. This reduction in UDS is more than can be explained by the completion of repair of X-ray-induced DNA lesions. We suggest that the reduction in UDS is the result of an X-ray-produced impairment of a least a part of the repair mechanism involved in correcting MMS-induced DNA lesions. When the time interval between a 600-R X-ray exposure and MMS treatment was between 3 and 20 h (latest time interval s;udied) there was a statistically significant reduction of UDS in the spermatids. No significant decrease in UDS response occurred when the time interval between radiation exposure and MMS treatment was less than approximately 3 h.     

The nucleotide sequence of cDNA coding for the structural proteins of foot-and-mouth disease virus

The complete nucleotide sequence of cDNA coding for the structural capsid polypeptides of foot-and-mouth disease virus (FMDV) (strain A(10)61) has been determined. Portions of the flanking sequence coding for the nonstructural proteins p20a and p52 are also provided. The three larger structural polypeptides VP1, VP2 and VP3 have unmodified Mrs of 23248, 24649 and 24213, respectively. The size of the smaller polypeptide, VP4, can only be estimated at 7360 because the 5'-limit of its coding region is not yet known with certainty. The sequence data for VP1 (the major immunising antigen) and the amino-terminal quarter of p52 are compared with the data of Kurz et al. (Nucl. Acids Res. 9 (1981) 1919-1931) for a different serotype (O1K). This shows that variation is much greater in the region coding for VP1 than in that coding for p52. This is reflected in the level of amino acid sequence variation predicted for the two proteins. Analysis of relative codon usage reveals a strong bias in favour of C and G over U and A in the third base position. The dinucleotide frequencies show a bias against A-U and U-A, and for A-C and C-A.     

Changes in the reactivity of proteins of rat liver ribosomes against [C]iodoacetamide depending on their organization in ribosomal subparticles, 80S ribosomes and on the attachment of poly-(U)

(1) The isolated mixtures of ribosomal proteins can be substituted by [¹⁴C]-iodoacetamide up to an average of about 2 equivalents per 20 000 dalton. The extent of substitution of single proteins measured after two-dimensional polyacrylamide gel electrophoresis shows that all proteins are reactive.

(2) Also in the subunits, all proteins are accessible to substitution. Compared with isolated proteins, however, the reactivity is decreased and the amount of labelling for most proteins ranges as low as 5 to 20%.

(3) Reassociation of ribosomal subunits decreases the reactivity of 12 proteins of the small subunit and that of 20 proteins of the large subunit.

(4) The presence of messenger inhibits the substitution of 10 proteins of the small subunit and of 6 proteins of the large one.

(5) Seven proteins of the small subunit and 3 proteins of the large one are influenced both by the other subunit and by messenger-RNA.     

Estrogen regulation of superoxide dismutase in normal rat mammary tissues and mammary tumors

Multiple electrophoretic molecular variants of superoxide dismutase were demonstrated in normal rat mammary tissues and DMBA-induced rat mammary tumors. The specific activities of $\text{Cu}\text{Zu}$ superoxide dismutase in mammary tumors of estrogen-treated rats were not significantly different from those activities seen in normal rat mammary tissues. However, the enzyme activities of mammary tumors from untreated rats (no estrogen) were significantly lower than the activities of normal rat mammary tissues. Exogenous estrogen appeared to raise superoxide dismutase levels in DMBA-induced rat mammary tumors to those levels seen in normal rat mammary tissues.     

Hydrogen peroxide involvement in the rhodanese inactivation by dithiothreitol.

The conditions required to obtain rhodanese inactivation in the presence of dithiothreitol indicate the involvement of hydrogen peroxide produced by metal-ion catalyzed oxidation of dithiothreitol. Inhibition of dithiothreitol oxidation by a chelating agent, or by removal of hydrogen peroxide by catalase prevents the enzyme inactivation. The inactivated enzyme contains a disulfide bond resulting from the oxidation of the catalytic sulfhydryl group and another sulfhydryl group close to it. This disulfide might be formed via a sulfenic intermediate.     

Analysis of sequence homology between human and mouse mitochondrial DNA

The base sequence homology between human and mouse mitochondrial DNA has been investigated by hybridization of highly labelled mitochondrial DNA probes with restriction fragments of mitochondrial DNA blotted according to the Southern technique. By this analysis, the homologous regions have been found to be widely distributed along the mitochondrial genome. Competition hybridization experiments with unlabelled HeLa mitochondrial RNAs have shown that most of the cross-hybridization involves the ribosomal and 4 S RNA genes.     

Proline hydroxylation by cell free extract of a streptomycete

Free L-proline was hydroxylated to free L-hydroxyproline by cell free extract of Streptomyces griseoviridus P8648. The hydroxylation reaction required ferrous ion, 2-ketoglutarate and ascorbate. Zinc ion, ethylenediaminetetraacetic acid and alpha,alpha'-dipyridyl inhibited the reaction. Optimum temperature and pH were 25.0 degrees C and 7.5, respectively.     

Purification and partial characterization of a 19kD/pI 4.5 nucleolar phosphoprotein

Two-dimensional PAGE analysis of proteins associated with the slowly sedimenting "fibrillar" structures of HeLa nucleoli revealed a protein with a M of 19,000 and a pI of 4.5 which was highly labeled both with 32P-orthophosphate and 35S-methionine. The protein was isolated from Novikoff hepatoma nucleoli by extraction in 0.35 M NaCl and 5 mM DTT followed by chromatography in EDTA on DEAE-cellulose and Sephadex G-100. The protein was homogeneous with respect to two-dimensional PAGE, number of tryptic peptides and carboxyl terminal analysis. The protein contained an acidic/basic amino acid ratio of 2.1, 7 residues of methionine, 2 residues of cysteine, a blocked amino terminus and a carboxyl terminal lysylleucine.