Different half-lives of messenger RNA corresponding to different segments of the tryptophan operon of Escherichia coli

In genetically derepressed strains (trpR⁻) of Escherichia coli which are growing exponentially, messenger RNA regions corresponding to different segments of the trp operon are labeled with different kinetics, suggesting that operator-proximal and distal regions of trp-mRNA have different half-lives. This conclusion was confirmed by direct measurement of trp-mRNA decay; the half-lives for different mRNA regions at 30 °C were found to be 60 seconds for trpE-mRNA, 75 seconds for trpDC-mRNA, and 95 to 115 seconds for trpBA-mRNA. Deletions of genetic segments within the operator-proximal region of the operon reduce the half-life of trp BA-mRNA. Large deletions which place the BA region near the operator reduce the half-life of trpBA-mRNA to values similar to that of trpE-mRNA in the parental strain. Therefore location in the message rather than primary structure appears to determine the half-life of each mRNA region. Several of the internal deletions have a polar effect on the synthesis of the trpB and trpA polypeptides. However, the reduction in trpBA-mRNA half-life does not appear to be due to polarity because trpBA-mRNA half-life is reduced to the same value in three deletion mutants in which there is a sevenfold difference in polarity. These results are compatible with a model of trp-mRNA degradation in which the initial degradative event occurs near the 5′ end of the mRNA molecule and is followed by over-all degradation in the 3′ direction, with random or non-random delays causing an increase in half-life of about 10% per 1000 nucleotides mRNA. Our findings are not compatible with a model of normal degradation in which the entire mRNA molecule is the target for the initial degradative event.     

Nucleotide sequence of the Neurospora crassa trp-3 gene encoding tryptophan synthetase and comparison of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae and Escherichia coli

The complete nucleotide sequence of the Neurospora crassa trp-3 gene-encoding tryptophan synthetase has been determined; we present an analysis of its structure. A comparison of the deduced amino acid sequence of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae (encoded by the TRP5 gene) and Escherichia coli (encoded by the trpA and trpB genes) shows that the A and B domains (amino acid segments homologous to the trpA and trpB polypeptides, respectively) of the N. crassa and yeast polypeptides are in the same order (NH2-A-B-COOH). This arrangement is the reverse of the gene order characteristic of all prokaryotes that have been examined. N. crassa tryptophan synthetase has strong homology to the yeast TRP5 polypeptide (A domains have 54% identity; B domains have 75% identity), and somewhat weaker homology to the E. coli trpA and trpB polypeptides (A domains have 31% identity; B domains have 50% identity). The two domains of the N. crassa polypeptide are linked by a connector of 54-amino acid residues that has less than 25% identity to the 45-residue connector of the yeast polypeptide, although secondary structure analysis predicts both connectors would be alpha-helical. In contrast to the yeast TRP5 gene, which has no introns, the trp-3 coding region is interrupted by two introns 77 and 71 nucleotides in length. Both introns are located near the 5'-end of the gene and therefore not near the segment encoding the connector.     

Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides

Background  

Lignin and hemicelluloses are the major components limiting enzyme infiltration into cell walls. Determination of the topochemical distribution of lignin and aromatics in sugar cane might provide important data on the recalcitrance of specific cells. We used cellular ultraviolet (UV) microspectrophotometry (UMSP) to topochemically detect lignin and hydroxycinnamic acids in individual fiber, vessel and parenchyma cell walls of untreated and chlorite-treated sugar cane. Internodes, presenting typical vascular bundles and sucrose-storing parenchyma cells, were divided into rind and pith fractions.     

Placentation in dolphins from the Amazon River Basin: the Boto, Inia geoffrensis, and the Tucuxi, Sotalia fluviatilis

A recent reassessment of the phylogenetic affinities of cetaceans makes it timely to compare their placentation with that of the artiodactyls. We studied the placentae of two sympatric species of dolphin from the Amazon River Basin, representing two distinct families. The umbilical cord branched to supply a bilobed allantoic sac. Small blood vessels and smooth muscle bundles were found within the stroma of the cord. Foci of squamous metaplasia occurred in the allanto-amnion and allantochorion. The interhemal membrane of the placenta was of the epitheliochorial type. Two different types of trophoblastic epithelium were seen. Most was of the simple columnar type and indented by fetal capillaries. However, there were also areolar regions with tall columnar trophoblast and these were more sparsely supplied with capillaries. The endometrium was well vascularised and richly supplied with actively secreting glands. These findings are consistent with the current view that Cetacea are nested within Artiodactyla as sister group to the hippopotamids.     

Amino-terminal sequences of indoleglycerol phosphate synthetase of Escherichia coli and Salmonella typhimurium.

The partial sequences of the first 40 residues of indoleglycerol phosphate synthetase of Escherichia coli and Salmonella typhimurium were determined, and three amino acid differences were observed among the 38 residues compared.     

Naturally occurring sites within the Shigella dysenteriae tryptophan operon severely limit tryptophan biosynthesis.

We investigated the structural, functional, and regulatory properties of the Shigella dysenteriae tryptophan (trp.) operon in transduction hybrids in which the cysB-trp-region of Escherichia coli is replaced by the corresponding region from S. dysenteriae. Tryptophan biosynthesis was largely blocked in the hybrids, although the order of the structural genes was identical with that of E. coli. Nutritional tests and enzyme assays revealed that the hybrids produced a defective anthranilate synthetase (ASase). Deletion mapping identified two distinct sites in trpE, each of which was partially responsible for the instability and low activity of ASase. We also discovered a pleiotropic site trpP (S) that maps outside the structural gene region and is closely linked to the S. dysenteriae trp operator. trpP (S) reduced the rate of trp messenger ribonucleic acid synthesis, and consequently trp enzyme levels, 10-fold relative to wild-type E. coli. In recombinants in which the structural genes of E coli were under the control of the S. dysenteriae promoter, enzyme levels were also reduced 10-fold. In some fast-growing revertants of the original hybrids, the rates of trp messenger ribonucleic acid synthesis and levels of tryptophan synthetase were restored to values characteristic of wild-type E.coli. Thus, the Trp auxotrophy associated with the S dysenteriae trp operon derives from the combination of a defective ASase and decreased expression of the entire operon imposed by trpP (S).