Half-lives of messenger RNA species during growth and differentiation of Dictyostelium discoideum

The half-lives of functional messenger RNAs were determined by a method employing the drugs actinomycin D and daunomycin for the inhibition of mRNA synthesis; the activity of extracted mRNAs was determined by an in vitro translation assay. Several controls indicated that this method yielded reliable values for mRNA half-lives; in particular, the declining rate of protein synthesis in the presence of the drugs is due predominantly to the decay of translatable mRNA. This method was used to determine the half-lives of two specific mRNAs—encoding actin and a protein of MW 51,000—as well as that of total cytoplasmic mRNA activity during growth and at several times in differentiation. The half-lives of at least these two mRNAs were shown to be distinctly different from that of the total mRNA population—about 4 hr. However, no significant change in any of these half-lives was observed between growing and developing cells. Therefore wholesale alterations in the degradation rates of total and at least specific messages do not appear to play a role in the regulation of gene expression during Dictyostelium development.     

Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene.

Mammalian ribonucleotide reductase consists of two non-identical subunits, proteins M1 and M2. M2-related DNA sequences are present on mouse chromosomes 4, 7, 12 and 13. However, M2-overproducing mouse cells show amplification of a chromosome 12-specific, single 13 kb HindIII fragment, which probably represents the active gene. We have isolated this fragment from parental mouse cell DNA and used it to clone and characterize the functional M2 gene. The 5770 bp transcribed M2 sequence contains ten exons separated by nine 95-917 bp introns. The 501 bp of 5' flanking DNA is G + C rich and contains TTTAAA and CCAAT sequences as well as potential Sp1 binding sites. The M2-related sequence on chromosome 13, which contains only the last six exons and several internal rearrangements, is a pseudogene. Transfection of BALB/3T3 cells with the M2 gene resulted in stable transformants with a 10-fold reduction in sensitivity to hydroxyurea, compared to control cells. This confirmed that the cloned M2 genomic DNA represents the functional gene and conclusively establishes the link between hydroxyurea resistance and M2 expression in mammalian cells. M2 genomic DNA should be a valuable dominant, selectable marker for identifying and isolating stable co-transformants.     

Axonal Transport, Deposition, and Metabolic Turnover of Glycoproteins in the Rat Optic Pathway

Abstract: Following intraocular injection of [³H]fucose in the rat, radioactive glycoproteins are rapidly transported to the nerve terminals in at least two waves, one with a peak at 8 h and a second with a peak at about a week. The molecular weight distribution of radioactive peptides in ach transport wave as determined by gel electrophoresis in buffers containing sodium dodecyl sulfate is very similar. Most of the many glycopeptides in the first wave of rapid transport pass through the optic tract in unison (apparent half-life of about 15 h) and are preferentially destined for the nerve endings. However, two proteins of apparent M. W. 28,000 and 49,000 are preferentially retained in the axons. The remaining proteins, after reaching the nerve endings (superior colliculus), decay with apparent half-lives ranging from 17 to 34 h. During the second wave a large amount of the 28,000 and 49,000 M. W. peptides are again preferentially retained in the axons. The remaining proteins, on reaching the nerve endings, decay with apparent half-lives ranging from 5 to 9 days. Subcellular fractionation of the superior colliculus supports the hypothesis that the 49,000 and 28,000 M. W. peptides are the predominantly labeled glycoproteins present in myelinated axons (representing over 50% of the radioactive glycoproteins 7 days following injection), although they are probably also present in membranes of the nerve endings. A comparison with glycoprotein transport in other tracts (geniculocortical and nigrostriatal tracts) suggests that glycoprotein transport in these pathways has many similarities to glycoprotein transport in the retinal ganglion cells, and that the optic system is a good general model for axonal transport in the CNS.     

Binding of IKe gene 5 protein to polynucleotides. Fluorescence binding experiments of IKe gene 5 protein and mutual cooperativity of IKe and M13 gene 5 proteins

Fluorescence studies of the binding of IKe gene 5 protein to various polynucleotides were performed to obtain insight into the question as to what extent the binding characteristics of the gene 5 proteins of the IKe and M13 phages resemble and/or differ from each other. The fluorescence of IKe gene 5 protein is quenched 60% upon binding to most polynucleotides. At moderate salt concentrations, i.e., below 1 M salt, the binding stoichiometry is 4.0 +/- 0.5 nucleotides per IKe gene 5 protein monomer. The affinity of the protein for homopolynucleotides depends strongly on sugar and base type; in order of increasing affinities we find poly(rC) less than poly(dA) less than poly(rA) less than poly(dI) less than poly(rU) less than poly(dU) less than poly(dT). For most polynucleotides studied, the affinity depends linearly on the salt concentration: [d log (Kint omega)]/(d log [M+]) = -3. The binding is highly cooperative. The cooperativity parameter omega, as deduced from protein titration curves, is 300 +/- 150 and appears independent of the type of polynucleotide studied. Estimation of this binding parameter from salt titrations of gene 5 protein-polynucleotide complexes results in systematically higher values. A comparison of the binding data of the IKe and M13 gene 5 proteins shows that the fluorescence quenching, stoichiometry, order of binding affinities, and cooperativity in the binding are similar for both proteins. From this it is concluded that at least the DNA binding grooves of both proteins must show a close resemblance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complex of bacteriophage M13 single-stranded DNA and gene 5 protein

Lysates of bacteriophage M13-infected cells contain numerous unbranched filamentous structures approximately 1·1 μm long × 160 Å wide, that is, slightly longer and considerably wider than M13 virions. These structures are complexes of viral single-stranded DNA molecules with M13 gene 5 protein, a non-capsid protein required for single-stranded DNA production. All, or nearly all, of the single-stranded DNA from the infected cells and at least half to two-thirds of the gene 5 protein molecules are found as complex in the lysates. The complex contains about 1300 gene 5 protein molecules per DNA molecule but little if any of the two known capsid proteins. The complex is much less stable than virions in the presence of salt or ionic detergent solutions and in electron micrographs it appears to have a much looser and more open structure. If an excess of M13 single-stranded DNA is added to complex in a lysate, the gene 5 protein molecules from the complex redistribute onto all of the added as well as the original DNA, again suggesting a rather loose association of protein and DNA.By electron microscopy, the complex from infected cells appears to differ structurally from complex formed in vitro between purified single-stranded DNA and purified gene 5 protein. Because of this apparent structural difference and because previous experiments suggested the presence of complex in vivo, we presume that the complex which we have found in lysates of infected cells previously did exist as such inside the cells, but we have been unable to exclude that it formed during or after lysis. If it is assumed that complex does occur in vivo, the results of pulse-chase radioactive labeling experiments on infected cells can be interpreted as showing that with time the single-stranded DNA leaves complex, presumably to be matured into virions, while the gene 5 protein molecules are re-used to form more complex.     

Sequence and cloning of bacteriophage T4 gene 63 encoding RNA ligase and tail fibre attachment activities

The sequence of gene 63 of bacteriophage T4 was determined by a shotgun approach. Small DNA fragments, derived by sonication of a restriction fragment that encompasses the region of gene 63, were cloned in M13 vectors and sequenced by the 'dideoxy' method. The position of the gene was established by comparison with the sequence of a gene 63 amber mutant. Knowledge of the DNA sequence of gene 63 and surrounding regions has allowed the construction of a clone of gene 63 in which RNA ligase production is under the control of the lac promoter of bacteriophage M13mp8. Infected E. coli cells can be induced to produce a protein indistinguishable from commercially available RNA ligase.     

METABOLISM OF ARACHIDONOYL PHOSPHOGLYCERIDES IN MOUSE BRAIN SUBCELLULAR FRACTIONS

Abstract— Mouse brain subcellular fractions were prepared at 1, 12, and 24 h and 3 and 8 days after intracerebral injections of [1-¹⁴C]arachidonate. Initially, radioactivity was mainly distributed in the microsomal and synaptosomal fractions, but the proportion of radioactivity in the myelin increased from 5 to 16% within 8 days. Radioactivity of the microsomal lipids started to decline at 1 h after injection, and the decay was represented by two pools with half-lives of 19 h and 10 days, respectively. Radioactivity in the synaptosomal and myelin fractions did not reach a maximum until 24 h after injections. The half-life for turnover of synaptosomal lipids was 9 days.
The decline of radioactivity measured in the microsomal fraction was due mainly to diacyl-GPC and diacyl-GPI, since radioactivity of other phosphoglycerides (diacyl-GPS, diacyl-GPE and alkenyl-acyl-GPE) continued to increase for 12-24 h. In this fraction, half-lives of 10-14 h were obtained for the fast turnover pools of diacyl-GPC and diacyl-GPI, and slow turnover pools with half-lives of 7 days for diacyl-GPI and 10-14 days for other phosphoglycerides were also present. Among the synaptosomal phosphoglycerides, radioactivity of diacyl-GPI declined in a biphasic mode, thus exhibiting half-lives of 5 h and 5 days. Incorporation of labelled arachidonate into diacyl-GPE and diacyl-GPS in the synaptosomal fractions was observed for a period of 24 h. The half-lives for these phosphoglycerides ranged from 8 to 12 days. Results of the study have demonstrated the presence of small pools of arachidonoyl-GPI in synaptosomal and microsomal fractions which were metabolically more active than other arachidonoyl containing phosphoglycerides.     

人溶菌酶基因的合成和克隆

用固相亚磷酰胺法合成了人溶菌酶的全基因,全长为409bp,它包括了编码人溶菌酶的结梅基因,起始密码于ATG,终止密码子TAA、TGA,以及两端的BamHI和SphI的识别顺序。整个基因分成24个寡聚核苷酸片段进行合成,每个片段长度分别为26至38个核苷酸．然后用两种方法酶促连接成完整的人溶菌酶基因。基因克隆到M13载体上。用点杂交和限制酶酶切分析确定阳性克隆株。用双脱氧链终止法进行序列分析,证实所合成的人溶菌酶基因序列与设计的完全一致。