(1/3129) The role of oocyte transcription, the 5'UTR, and translation repression and derepression in Drosophila gurken mRNA and protein localization.
The establishment of the major body axes of the Drosophila egg and future embryo requires strict regulation of gurken mRNA and protein localization. Here, we show that grk mRNA and protein localization is dependent on synthesis of grk transcripts in the oocyte nucleus and on RNA localization elements in the 5' portion of the transcript. We also show that gurken mRNA and protein localization is dependent on region-specific translation of gurken transcripts and identify K10 as a probable negative regulator of gurken translation. (+info
(2/3129) Comparison of Bombyx mori and Helicoverpa armigera cytoplasmic actin genes provides clues to the evolution of actin genes in insects.
The cytoplasmic actin genes BmA3 and BmA4 of Bombyx mori were found clustered in a single genomic clone in the same orientation. As a similar clustering of the two cytoplasmic actin genes Ha3a and Ha3b also occurs in another lepidopteran, Helicoverpa armigera, we analyzed the sequence of the pair of genes from each species. Due to the high conservation of cytoplasmic actins, the coding sequence of the four genes was easily aligned, allowing the detection of similarities in noncoding exon and intron sequences as well as in flanking sequences. All four genes exhibited a conserved intron inserted in codon 117, an original position not encountered in other species. It can thus be postulated that all of these genes derived from a common ancestral gene carrying this intron after a single event of insertion. The comparison of the four genes revealed that the genes of B. mori and H. armigera are related in two different ways: the coding sequence and the intron that interrupts it are more similar between paralogous genes within each species than between orthologous genes of the two species. In contrast, the other (noncoding) regions exhibited the greatest similarity between a gene of one species and a gene of the other species, defining two pairs of orthologous genes, BmA3 and HaA3a on one hand and BmA4 and HaA3b on the other. However, in each species, the very high similarities of the coding sequence and of the single intron that interrupts it strongly suggest that gene conversion events have homogenized this part of the sequence. As the divergence of the B. mori genes was higher than that of the H. armigera genes, we postulated that the gene conversion occurred earlier in the B. mori lineage. This leads us to hypothesize that gene conversion could also be responsible for the original transfer of the common intron to the second gene copy before the divergence of the B. mori and H. armigera lineages. (+info
(3/3129) unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA.
Initiation of translation of the animal picornavirus RNAs occurs via a mechanism of direct ribosome entry, which requires a segment of the 5' UTR of the RNA, known as the internal ribosome entry site (IRES). In addition, translation of the enterovirus and rhinovirus (HRV) subgroups requires cellular trans-acting factors that are absent from, or limiting in rabbit reticulocytes, but are more abundant in HeLa cell extracts. It has been shown previously that HeLa cells contain two separable activities, each of which independently stimulates HRV IRES-dependent translation when used to supplement reticulocyte lysate; one of these activities was identified as polypyrimidine tract-binding protein (PTB). Here, the purification of the second activity is achieved by use of an RNA-affinity column based on the HRV 5' UTR. It comprises two components: a 38-kD protein (p38), which is a novel member of the GH-WD repeat protein family and has no intrinsic RNA-binding activity; and a 96- to 97-kD protein doublet, which was identified as unr, an RNA-binding protein with five cold-shock domains. Coimmunoprecipitation with antibodies against either protein shows that the two proteins interact with each other, and thus p38 is named unrip (unr-interacting protein). Recombinant unr acts synergistically with recombinant PTB to stimulate translation dependent on the rhinovirus IRES. In contrast, unr did not significantly augment the PTB-dependent stimulation of poliovirus IRES activity. (+info
(4/3129) The Bradyrhizobium japonicum nolA gene encodes three functionally distinct proteins.
Examination of nolA revealed that NolA can be uniquely translated from three ATG start codons. Translation from the first ATG (ATG1) predicts a protein (NolA1) having an N-terminal, helix-turn-helix DNA-binding motif similar to the DNA-binding domains of the MerR-type regulatory proteins. Translation from ATG2 and ATG3 would give the N-terminally truncated proteins NolA2 and NolA3, respectively, lacking the DNA-binding domain. Consistent with this, immunoblot analyses of Bradyrhizobium japonicum extracts with a polyclonal antiserum to NolA revealed three distinct polypeptides whose molecular weights were consistent with translation of nolA from the three ATG initiation sites. Site-directed mutagenesis was used to produce derivatives of nolA in which ATG start sites were sequentially deleted. Immunoblots revealed a corresponding absence of the polypeptide whose ATG start site was removed. Translational fusions of the nolA mutants to a promoterless lacZ yielded functional fusion proteins in both Escherichia coli and B. japonicum. Expression of NolA is inducible upon addition of extracts from 5-day-old etiolated soybean seedlings but is not inducible by genistein, a known inducer of the B. japonicum nod genes. The expression of both NolA2 and NolA3 requires the presence of NolA1. NolA1 or NolA3 is required for the genotype-specific nodulation of soybean genotype PI 377578. (+info
(5/3129) CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock.
Escherichia coli contains the CspA family, consisting of nine proteins (CspA to CspI), in which CspA, CspB, and CspG have been shown to be cold shock inducible and CspD has been shown to be stationary-phase inducible. The cspI gene is located at 35.2 min on the E. coli chromosome map, and CspI shows 70, 70, and 79% identity to CspA, CspB, and CspG, respectively. Analyses of cspI-lacZ fusion constructs and the cspI mRNA revealed that cspI is cold shock inducible. The 5'-untranslated region of the cspI mRNA consists of 145 bases and causes a negative effect on cspI expression at 37 degrees C. The cspI mRNA was very unstable at 37 degrees C but was stabilized upon cold shock. Analyses of the CspI protein on two-dimensional gel electrophoresis revealed that CspI production is maximal at or below 15 degrees C. Taking these results together, E. coli possesses a total of four cold shock-inducible proteins in the CspA family. Interestingly, the optimal temperature ranges for their induction are different: CspA induction occurs over the broadest temperature range (30 to 10 degrees C), CspI induction occurs over the narrowest and lowest temperature range (15 to 10 degrees C), and CspB and CspG occurs at temperatures between the above extremes (20 to 10 degrees C). (+info
(6/3129) Structural characterization of the gene for human histidine-rich glycoprotein, reinvestigation of the 5'-terminal region of cDNA and a search for the liver specific promoter in the gene.
Genomic DNA libraries were screened for the human histidine-rich glycoprotein (HRG) gene and a sequence of 15,499 nucleotides was determined. The gene is composed of 7 exons and 6 introns, and all the exon-intron boundaries match the consensus GT/AG sequence for donor and acceptor splice sites. Each of cystatin-like domains I and II of HRG is encoded by three exons, exons I to III and exons IV to VI, respectively, like those of other members of the cystatin superfamily. The entire C-terminal half of the molecule is encoded by the largest exon, VII. The first 103 nucleotides of the cDNA sequence reported for human HRG [Koide, T., Foster, D., Yoshitake, S. , and Davie, E.W. (1986) Biochemistry 25, 2220-2225] could not be found in the determined gene sequence. A homology search of this sequence against a database showed the complete matching to a part of the yeast mitochondrial DNA encoding 21S ribosomal RNA. Rapid amplification of cDNA 5' ends (5'-RACE) analysis revealed that the cDNA has multiple 5'-ends and that a possible starting point is nucleotide 104 of the reported cDNA sequence. These results suggest that the first 103 nucleotides of the cDNA sequence reported for human HRG originated from yeast mitochondrial DNA and were incidentally incorporated into the HRG cDNA in the process of the construction of a cDNA library. Various fragments obtained on restriction endonuclease digestion of the 5'-noncoding region of the HRG gene were ligated to the chloramphenicol acetyltransferase (CAT) gene and then transfected into HepG2 and 293 cells to analyze the promoter activity. The sequence between -262 and -21 from the putative translation initiation site supported the expression of CAT in HepG2 cells but not in 293 cells, suggesting that this segment promotes the liver-specific transcription of the human HRG gene. (+info
(7/3129) A multisubunit acetyl coenzyme A carboxylase from soybean.
A multisubunit form of acetyl coenzyme A (CoA) carboxylase (ACCase) from soybean (Glycine max) was characterized. The enzyme catalyzes the formation of malonyl CoA from acetyl CoA, a rate-limiting step in fatty acid biosynthesis. The four known components that constitute plastid ACCase are biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and the alpha- and beta-subunits of carboxyltransferase (alpha- and beta-CT). At least three different cDNAs were isolated from germinating soybean seeds that encode BC, two that encode BCCP, and four that encode alpha-CT. Whereas BC, BCCP, and alpha-CT are products of nuclear genes, the DNA that encodes soybean beta-CT is located in chloroplasts. Translation products from cDNAs for BC, BCCP, and alpha-CT were imported into isolated pea (Pisum sativum) chloroplasts and became integrated into ACCase. Edman microsequence analysis of the subunits after import permitted the identification of the amino-terminal sequence of the mature protein after removal of the transit sequences. Antibodies specific for each of the chloroplast ACCase subunits were generated against products from the cDNAs expressed in bacteria. The antibodies permitted components of ACCase to be followed during fractionation of the chloroplast stroma. Even in the presence of 0.5 M KCl, a complex that contained BC plus BCCP emerged from Sephacryl 400 with an apparent molecular mass greater than about 800 kD. A second complex, which contained alpha- and beta-CT, was also recovered from the column, and it had an apparent molecular mass of greater than about 600 kD. By mixing the two complexes together at appropriate ratios, ACCase enzymatic activity was restored. Even higher ACCase activities were recovered by mixing complexes from pea and soybean. The results demonstrate that the active form of ACCase can be reassembled and that it could form a high-molecular-mass complex. (+info
(8/3129) Sequence heterogeneity within three different regions of the hepatitis G virus genome.
Two sets of primers derived from the 5'-terminal region and the NS5 region of the hepatitis G virus (HGV) genome were used to amplify PCR fragments from serum specimens obtained from different parts of the world. All PCR fragments from the 5'-terminal region (5'-PCR, n = 56) and from the NS5 region (NS5-PCR, n = 85) were sequenced and compared to corresponding published HGV sequences. The range of nucleotide sequence similarity varied from 74 and 78% to 100% for 5'-PCR and NS5-PCR fragments, respectively. Additionally, five overlapping PCR fragments comprising an approximately 2.0-kb structural region of the HGV genome were sequenced from each of five sera obtained from three United States residents. These sequences were compared to 20 published sequences comprising the same region of the HGV genome. Nucleotide and deduced amino acid sequences obtained from different individuals were homologous from 82.9 to 93. 6% and from 90.4 to 99.0%, respectively. Sequences obtained from follow-up specimens were almost identical. Comparative analysis of deduced amino acid sequences of the HGV structural proteins and hepatitis C virus (HCV) structural proteins combined with an analysis of predicted secondary structures and hydrophobic profiles allowed prediction of processing sites within the HGV structural proteins. A phylogenetic sequence analysis performed on the 2.0-kb structural region supports the existence of three previously identified HGV genetic groups. However, phylogenetic analysis performed on only small DNA fragments yielded inconsistent genetic grouping and failed to confirm the existence of genetic groups. Thus, in contrast to HCV where almost any region can be used for genotyping, only large or carefully selected genome fragments can be used to identify consistent HGV genetic groups. (+info