The mechanism of the hyperglycaemic action of synthetic peptides related to the C-terminal sequence of human growth hormone

Synthetic part sequences of human pituitary growth hormone (hGH 176–191 and hGH 177–191) corresponding to residues 176–191 or 177–191 of the hormone have been tested for their effects on glycogen and pyruvate metabolism in the rat, both in vivo and in vitro. When injected, the peptides caused transient increases in blood glucose and lactate, while decreasing the activity ratio of glycogen synthase in muscle, adipose tissue and liver and of pyruvate dehydrogenase in muscle and adipose tissue, but not in liver. These decreases were associated with the conversion of the enzymes from their active to their inactive forms, since the peptides did not affect the total amount of either the synthase or the dehydrogenase. The time course of the effect on the enzymes was similar to that for the effect on blood metabolites, and responses for synthase were produced over the range 0.07–7 nmols hGH 177–191/kg body weight. Phosphorylase activity was not affected by the peptides, nor was the capacity to dispose of injected L-lactate. Experiments with adipocytes and hepatocytes showed that the peptides also affected glycogen synthase and pyruvate dehydrogenase activities in vitro. The peptides had no effect on the overall rate of gluconeogenesis from lactate by hepatocytes. However, at times corresponding to those at which glycogen synthase was inactivated, the peptides caused increased incorporation of lactate into free glucose and decreased incorporation into glycogen. It was concluded that the peptides acted directly on their target tissues, and that the observed hyperlactataemia was the result of the inactivation of pyruvate dehydrogenase. The addition lactate increased the flux through the gluconeogenic pathway, and appeared as glucose because the peptide also inactivated glycogen synthase. Thus, the hyperglycaemia produced by hGH 177–199 and related peptides is explicable in terms of a modified Cori Cycle.     

Targeted integration of foreign genes into repetitive sequences of the Brevibacterium lactofermentum chromosome

Abstract The nucleotide sequence of a gene coding for a 37 kDa subunit of a cytosolic malate dehydrogenase of Trichomonas vaginalis was established. The sequences of a gDNA clone and a cDNA clone, which lacked seven amino-terminal codons, were identical, indicating an absence of introns from the gene. Cell fractionation combined with sequencing of peptide fragments of the purified enzyme showed that the gene codes for an expressed cytosolic enzyme. The derived amino acid sequence was closely related to cytosolic malate dehydrogenases from animals and plants and from the eubacteria Thermus aquaticus and Mycobacterium leprae and was more distant from the enzyme of mitochondria and from Escherichia coli and certain other eubacteria. In phylogenetic reconstructions this enzyme shared a most recent common ancestor with other cytosolic enzymes.     

A second‐generation genetic linkage map for bighead carp (Aristichthys nobilis) based on microsatellite markers

Bighead carp (Aristichthys nobilis) is an important aquaculture fish worldwide. Genetic linkage maps for the species were previously reported, but map resolution remained to be improved. In this study, a second‐generation genetic linkage map was constructed for bighead carp through a pseudo‐testcross strategy using interspecific hybrids between bighead carp and silver carp. Of the 754 microsatellites genotyped in two interspecific mapping families (with 77 progenies for each family), 659 markers were assigned to 24 linkage groups, which were equal to the chromosome numbers of the haploid genome. The consensus map spanned 1917.3 cM covering 92.8% of the estimated bighead carp genome with an average marker interval of 2.9 cM. The length of linkage groups ranged from 52.2 to 133.5 cM with an average of 79.9 cM. The number of markers per linkage group varied from 11 to 55 with an average of 27.5 per linkage group. Normality tests on interval distances of the map showed a non‐normal marker distribution; however, significant correlation was found between the length of linkage group and the number of markers below the 0.01 significance level (two‐tailed). The length of the female map was 1.12 times that of the male map, and the average recombination ratio of female to male was 1.10:1. Visual inspection showed that distorted markers gathered in some linkage groups and in certain regions of the male and female maps. This well‐defined genetic linkage map will provide a basic framework for further genome mapping of quantitative traits, comparative mapping and marker‐assisted breeding in bighead carp.     

Synthetic DNA probes for the identification of sibling species in the Anopheles gambiae complex

The cloned DNA sequences pAna1, pAnq1 and pAnm14, which may be used to distinguish between at least five of the six species in the Anopheles gambiae Giles complex of Afrotropical malaria vector mosquitoes, have been sequenced. Each clone was found to possess a series of repeated sequences of 41, 30 and 163 bases respectively. In pAnq1 and pAnm14 the repeats were in direct tandem array, whilst in pAna1 the repetitive sequence was found to be interspersed by 15-17 variable bases. A comparison of a number of copies of each of the repetitive sequences within the three clones enabled the definition of the consensus sequence for each repetitive element. Based on these consensus sequences, three oligonucleotides of 21, 23 and 26 bases were derived from pAna1, pAnq1 and pAnm14 respectively. When tested as probes against DNA dot-blots and squash-blots of mosquito specimens, each oligonucleotide retained the same species-specificity as the original clones from which they were derived. The radioactively labelled oligonucleotides were able to detect as little as 5 ng of target genomic DNA in an overnight autoradiographic exposure. The synthetic DNA probes will form the basis of a simplified system for the field identification of Anopheles gambiae sibling species specimens.     

Trichoderma reesei sequences that bind to the nuclear matrix enhance transformation frequency

Three DNA fragments, trs1, 2 and 3, were isolated from the Trichoderma reesei genome on the basis of their ability to promote autonomous replication of plasmids in Saccharomyces cerevisiae. Each trs element bound specifically to the isolated T. reesei nuclear matrix in vitro, and two of them bound in vivo, indicating that they are matrix attachment regions (MARs). A similar sequence previously isolated from Aspergillus nidulans (ans1) was also shown to bind specifically to the T. reesei nuclear matrix in vitro. The T. reesei MARs are AT-rich sequences containing 70%, 86% and 73% A+T over 2.9, 0.8 and 3.7 kb, respectively for trs1, 2 and 3. They exhibited no significant sequence homology, but were shown to contain a number of sequence motifs that occur frequently in many MARs identified in other eukaryotes. However, these motifs occurred as frequently in the trs elements as in randomly generated sequences with the same A+T content. trs1 and 3 were shown to be present as single copies in the T. reesei genome. The presence of the trs elements in transforming plasmids enhanced the frequency of integrative transformation of T. reesei up to five fold over plasmids without a trs. No evidence was obtained to suggest that the trs elements promoted efficient replication of plasmids in T. reseei. A mechanism for the enhancement of transformation frequency by the trs elements is proposed. Received: 1 March 1997 / Accepted: 13 May 1997     

Evidence that the novel microorganism 'Z' may belong to a new genus in the family Chlamydiaceae

Abstract The purpose of this study was to investigate possible phylogenetic relationships of a new microorganism called 'Z'. The organism was previously shown to be similar to chlamydia in its growth cycle and some of its metabolic requirements, but different in others and in its major outer membrane protein. In this study we report the sequencing of 'Z's 16S ribosomal DNA and comparison of the sequence with that of other microorganisms, including chlamydia and rickettsiae. While chlamydial species have 95.5% sequence identity among themselves, 'Z' had 83% identity with them, and 73% identity with certain rickettsia-like organisms. Based on the sequence analyses and taking into account physiologic considerations, we believe that 'Z' may belong to a novel genus in the family Chlamydiaceae.