Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing.

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.     

The testis-specific phosphoglycerate kinase gene pgk-2 is a recruited retroposon.

In both humans and mice, two genes encode phosphoglycerate kinase, a key enzyme in the glycolytic pathway. The pgk-1 gene is expressed in all somatic cells, is located on the X chromosome, and contains 10 introns. The pgk-2 gene is expressed only in sperm cells, is located on an autosome, and has no introns. The nucleotide sequence of the pgk-2 gene suggests that it arose from pgk-1 more than 100 million years ago by RNA-mediated gene duplication. The pgk-2 gene may, then, be a transcribed retroposon. Thus, gene duplication by retroposition may have been used as a mechanism for evolutionary diversification.     

Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins.

In the eucaryotic nucleus, heterogeneous nuclear RNAs exist in a complex with a specific set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). The C proteins, C1 and C2, are major constituents of hnRNPs and appear to play a role in RNA splicing as suggested by antibody inhibition and immunodepletion experiments. With the use of a previously described partial cDNA clone as a hybridization probe, full-length cDNAs for the human C proteins were isolated. All of the cDNAs isolated hybridized to two poly(A)+ RNAs of 1.9 and 1.4 kilobases (kb). DNA sequencing of a cDNA clone for the 1.9-kb mRNA (pHC12) revealed a single open reading frame of 290 amino acids coding for a protein of 31,931 daltons and two polyadenylation signals, AAUAAA, approximately 400 base pairs apart in the 3' untranslated region of the mRNA. DNA sequencing of a clone corresponding to the 1.4-kb mRNA (pHC5) indicated that the sequence of this mRNA is identical to that of the 1.9-kb mRNA up to the first polyadenylation signal which it uses. Both mRNAs therefore have the same coding capacity and are probably transcribed from a single gene. Translation in vitro of the 1.9-kb mRNA selected by hybridization with a 3'-end subfragment of pHC12 demonstrated that it by itself can direct the synthesis of both C1 and C2. The difference between the C1 and C2 proteins which results in their electrophoretic separation is not known, but most likely one of them is generated from the other posttranslationally. Since several hnRNP proteins appeared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as multiple antigenically related polypeptides, this raises the possibility that some of these other groups of hnRNP proteins are also each produced from a single mRNA. The predicted amino acid sequence of the protein indicates that it is composed of two distinct domains: an amino terminus that contains what we have recently described as a RNP consensus sequence, which is the putative RNA-binding site, and a carboxy terminus that is very negatively charged, contains no aromatic amino acids or prolines, and contains a putative nucleoside triphosphate-binding fold, as well as a phosphorylation site for casein kinase type II. The RNP consensus sequence was also found in the yeast poly(A)-binding protein (PABP), the heterogeneous nuclear RNA-binding proteins A1 and A2, and the pre-rRNA binding protein C23. All of these proteins are also composed of at least two distinct domains: an amino terminus, which possesses one or more RNP consensus sequences, and a carboxy terminus, which is unique to each protein, being very acidic in the C proteins and rich in glycine in A1, and C23 and rich in proline in the poly(A)-binding protein. These findings suggest that the amino terminus of these proteins possesses a highly conserved RNA-binding domain, whereas the carboxy terminus contains a region essential to the unique function and interactions of each of the RNA-binding proteins.     

Rhythmical jaw movements and lateral ponto-medullary reticular neurons in rats

Neuronal activity in the lateral reticular formation was investigated in urethane-anesthetized rats. Stimulation of anterior and posterior cortical areas induced two types of rhythmical jaw movements (RJM). The effects of stimulation of these cortical areas, the peripheral nerves, and the trigeminal motor nucleus on these neurons and their activity during the RJM were analyzed. The smallest percentage of neurons receiving anterior cortical input received peripheral input, and most neurons with posterior cortical input received peripheral input. Sixty per cent of reticular neurons showed the rhythmical firing closely related to the RJM. Therefore, these neurons may participate in masticatory pattern formation.     

Esterogenic effect of zearalenone on the uterine acetyl cholinesterase in female rats

A single dose of zearalenone (10 g/g LBW) was injected intraperitoneally to Wistar Albino rats at the age of 50–100 days. The uterine acetyl cholinesterase enzyme was significantly increased in the treated animals compared to that in the controls. Therefore, zearalenone would be considered as an esterogenic effector for increasing the uterine acetyl cholinesterase which enhances uterine relaxation and subsequently improves its function for pregnancy in prematured-animals.Unlike estradiol, it was interesting to find that the estrogenicity of zearalenone was increased by the moderating progesterone hormone. Moreover, it was revealed in this study that the injected dose of zearalenone had no deleterious effects on the pregnant rats at 10–12 days period of gestation. The harmful effects of zearalenone on pregnant animals cited in the literature (11, 13, 19) were reviewed.     

Puromycin enhances 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster

The effect of cycloheximide and puromycin on 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster has been studied by one-dimensional and two-dimensional SDS polyacrylamide electrophoresis. It is found that puromycin, but not cycloheximide, when applied simultaneously with the hormone enhanced the hormone-induced synthesis of the early and late proteins. However, when puromycin was applied after hormone treatment, only the late proteins were induced. The possible implication of these observations is discussed.