Regulation of enzymes involved in ornithine/arginine metabolism in the parasitic trypanosomatid Herpetomonas samuelpessoai

Summary The genetic regulation of enzymes involved in arginine and ornithine synthesis has been investigated in the parasitic trypanosomatid Herpetomonas samuelpessoai. The activities of two enzymes involved in arginine synthesis, ornithine carbamoyltransferase (OCTase) and argininosuccinate lyase (ASLase) were depressed whereas the enzyme citrulline hydrolase (CHase), which is involved in ornithine synthesis, was increased in arginine supplemented cultures of the parasites. The depression of OCTase activity in the presence of arginine was not due to feedback inhibition and CHase activity of uninduced cultures was not enhanced by exogeneous arginine. Studies of the kinetics of OCTase induction and repression revealed that arginine blocks OCTase synthesis but does not cause destruction of the enzyme. Ornithine, but not citrulline. was found to counteract the arginine mediated repression of OCTase. Two classes of canavanine resistant mutants of H. samuelpessoai were isolated. One class was defective in arginine uptake whereas the other was affected in regulation of OCTase and ASLase which appear to be under coordinate control in H. samuelpessoai.     

Ovulation rate, embryo survival and ovarian sensitivity to gonadotrophins in mice selected for litter size and body weight

Single trait selection of mice for either large body size or large litter size resulted in an increased ovulation rate because of possible enhanced ovarian sensitivity to gonadotrophins. There was no difference in pre-implantation embryonic survival in either of the selected lines when compared to control mice. Selection for body weight did not alter post-implantation embryo survival, but fewer fetuses were lost after implantation in the litter size line compared to the control line. Index selection for large body size and small litter size did not change ovulation rate but increased pre- and post-implantation embryonic mortality. Selection for small body size and large litter size increased ovulation rate and decreased early embryonic death.     

The trypanosome VSG expression site encodes adenylate cyclase and a leucine-rich putative regulatory gene.

African trypanosomes are protozoan parasites that evade the host immune system by varying their dense antigenic coat. The Variant Surface Glycoprotein (VSG) is expressed exclusively from telomere-linked expression sites that contain in addition to the VSG gene, a number of open reading frames termed Expression Site Associated Genes (ESAGs). Here we demonstrate by complementation of a yeast mutant deleted for adenylate cyclase (cyr-1), that an ESAG from Trypanosoma equiperdum encodes an adenylate cyclase. Furthermore, we report that adjacent to adenylate cyclase in the expression site, is a separate open reading frame that encodes a protein sequence motif similar to the leucine-rich repeat regulatory domain of Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylate cyclases. The finding of two adjacent open reading frames homologous to a single enzyme in yeast suggests that the two expression site encoded proteins may interact to regulate adenylate cyclase activity during the course of an infection.     

Amino acid sequence diversity in mouse lambda 2 variable regions

The lambda-chains of immunoglobulins from BALB/c mice constitute the simplest system presently available for studying patterns of variable-region diversity. The limited number of V lambda and J lambda germ-line gene segments facilitates comparison of expressed and germ-line sequences. We report here the complete amino acid sequence of the variable regions of three lambda 2 chains and of one chain representing a V lambda 2----J lambda 3 rearrangement. Together with the previously determined sequence of the lambda 2 chain from myeloma MOPC-315, the results illustrate the following types of variable-region diversification: expression of a single V gene segment with more than one J segment, variability at the V-J junction, and presumably, somatic mutation in V and in J. The extent of somatic diversification in these lambda 2 chains is limited, consistent with results obtained previously with lambda 1 chains.     

Relationship of brown adipose tissue with growth and obesity differences in genetically selected mouse lines

A recent hypothesis considers brown adipose tissue (BAT) to be an important source of diet-induced thermogenesis (DIT). In turn, DIT and thermogenesis in general are believed to be key factors in the control of obesity of laboratory rodents. This hypothesis was developed from the study of single gene mutant obese rodents. The present research tested this hypothesis in mice with polygenic control of growth and obesity, which is more characteristic of the type of genetic variation expected in human and other mammalian populations. Control and high fat diets were used to test responses of five genetically selected lines of mice showing different patterns of growth and obesity. All lines deposited more fat on the high fat diet, but the most obese line showed the largest increase in BAT and the lipid-free dry (LFD) component of BAT. Use of LFD per unit body weight gave results which supported the hypothesis being tested, but it was argued that this measure is misleading. When brown and white adipose tissue growth relative to body weight were examined, 2 of the 10 line-diet groups showed alterations in BAT growth patterns. However, it was concluded that BAT, if involved at all, was not a major factor in growth and obesity differences.     

On the role of repeated sequences 5' to variant surface glycoprotein genes in African trypanosomes

In African trypanosomes, the DNA region situated upstream from all active and some silent variant surface glycoprotein genes (VSG genes) has a repetitive structure. This region is composed of a variable number of tandem repeats of an A + T-rich sequence which lacks the recognition sites for most commonly used restriction endonucleases, and is thus called 'barren region'. The length of the barren regions varies in different trypanosome variants from 0.2 to many kb. We have characterized the barren region upstream from the active VSG gene in two independent Trypanosoma equiperdum variants expressing the same VSG gene in the same expression site. To analyse the junction point between the expression site and the inserted gene, these two barren regions were cloned and sequenced. The longer barren region contains 14 repeats and the other contains two repeats. In both cases the junction point has been shown to lie within a repeat but different repeats were used in each case. These results argue that the repeats are important for the insertion of the duplicated-transposed gene into the expression site and that any repeat can be used.     

H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen

H-ras-transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form of 72 kDa, which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on its ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors, which has the same molecular mass and has been linked to their metastatic potential. Type IV collagenase consists of three domains. Two of them, the amino-terminal domain and the carboxyl-terminal domain, are homologous to interstitial collagenase and human and rat stromelysin. The middle domain, of 175 residues, is organized into three 58-residue head-to-tail repeats which are homologous to the type II motif of the collagen-binding domain of fibronectin. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin.