1.56 Å structure of mature truncated human fibroblast collagenase

The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 Å resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded β-sheet and three α-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase. © 1994 Wiley-Liss, Inc.     

Mapping of Murine Fibroblast Growth Factor Receptors Refines Regions of Homology between Mouse and Human Chromosomes

The genes for the fibroblast growth factor receptors Fgfr2, Fgfr3, and Fgfr4 have been mapped in the mouse using an interspecific backcross mapping panel. The Fgfr loci map to previously defined regions of homology between human and mouse chromosomes and provide additional information regarding human/mouse comparative mapping.     

Nuclear and chloroplast transformation inChlamydomonas reinhardtii: strategies for genetic manipulation and gene expression

Transformation of the nuclear, chloroplast, and mitochondrial genomes can now be accomplished inChlamydomonas reinhardtii. Many biosynthetic pathways are carried out in the chloroplast, and efforts to manipulate these pathways will require that gene products be directed to this compartment. Chloroplast proteins are encoded in either the chloroplast or nuclear genome. In the latter case they are synthesized in the cytoplasm and imported post-translationally into the chloroplast. Thus, strategies for expressing foreign genes or overexpressing endogenous genes whose products reside in the chloroplast could involve either genome. This paper reviews the present status of transformation methodology for the nuclear and chloroplast genomes inChlamydomonas. Considerations for expressing gene products in the chloroplast are discussed. Experimental evidence for homologous recombination during transformation of the nuclear genome is presented.     

Vector competence ofRhipicephalus sanguineus andDermacentor variabilis for American isolates ofBabesia gibsoni

To determine the identity of the tick vector of enzooticBabesia gibsoni in California, two common ixodid ticks were allowed to engorge uponB. gibsoni infected dogs. Sporozoites were observed in the salivary glands of prefed nymphalRhipicephalus sanguineus ticks that fed as larvae onB. gibsoni-infected dogs. A higher proportion (31%) of nymphal ticks that prefed on an uninfected dog for 48 hours contained sporozoites in their salivary glands than did ticks which had fed for 24 hours (13%). Sporozoites were not observed in the salivary glands of prefedR. sanguineus nymphs which were derived from the eggs of adult females that fed on an infected dog, in adults that were fed as nymph on an infected dog, or in the nymphal and adult uninfected controls.Dermacentor variabilis ticks appeared not to become infected. Although attempts to transmitB. gibsoni to susceptible, splenectomized dogs were unsuccessful,R. sanguineus would appear to be the most likely tick vector to maintain this piroplasm in California. This study was supported by grants from the Companion Animal Disease Laboratory, School of Veterinary Medicine, University of California, Davis.     

Demography and social structure of one group of muriquis (Brachyteles arachnoides)

We monitored one group of muriquis, or woolly spider monkeys (Brachyteles arachnoides), over a 9-year period at Fazenda Montes Claros, Minas Gerais, Brazil. The group grew from 22 to 42 individuals due to the births of 21 surviving infants. Eight immigrations involving immature females were offset by emigrations and disappearances. The home range of the group expanded as the group size increased. The group traveled as a cohesive unit during the first 6 years of the study, but recently it has begun to show greater tendencies to fission temporarily into smaller subgroups. Six adult males from the other muriqui group at this site have simultaneously increased their associations with the main study group. These observations indicate that the group is in a state of transition which may lead, ultimately, either to its division into two smaller units or to a more fluid social structure.     

Site-directed mutagenesis of the yeast PRP20/1SRM1 gene reveals distinct activity domains in the protein product

Prp20/Srm1, a homolog of the mammalian protein RCC1 in Saccharomyces cerevisiae, binds to double-stranded DNA (dsDNA) through a multicomponent complex in vitro. This dsDNA-binding capability of the Prp20 complex has been shown to be cell-cycle dependent; affinity for dsDNA is lost during DNA replication. By analyzing a number of temperature sensitive (ts) prp20 alleles produced in vivo and in vitro, as well as site-directed mutations in highly conserved positions in the imperfect repeats that make up the protein, we have determined a relationship between the residues at these positions, cell viability, and the dsDNA-binding abilities of the Prp20 complex. These data reveal that the essential residues for Prp20 function are located mainly in the second and the third repeats at the amino-terminus and the last two repeats, the seventh and eighth, at the carboxyl-terminus of Prp20. Carboxyl-terminal mutations in Prp20 differ from amino-terminal mutations in showing loss of dsDNA binding: their conditional lethal phenotype and the loss of dsDNA binding affinity are both suppressible by overproduction of Gsp1, a GTP-binding constituent of the Prp20 complex, homologous to the mammalian protein TC4/Ran. Although wild-type Prp20 does not bind to dsDNA on its own, two mutations in conserved residues were found that caused the isolated protein to bind dsDNA. These data imply that, in situ, the other components of the Prp20 complex regulate the conformation of Prp20 and thus its affinity for dsDNA. Gsp1 not only influences the dsDNA-binding ability of Prp20 but it also regulates other essential function(s) of the Prp20 complex. Overproduction of Gsp1 also suppresses the lethality of two conditional mutations in the penultimate carboxyl-terminal repeat of Prp20, even though these mutations do not eliminate the dsDNA binding activity of the Prp20 complex. Other site-directed mutants reveal that internal and carboxyl-terminal regions of Prp20 that lack homology to RCC1 are dispensable for dsDNA binding and growth.     

Reporter enzymes for the study of promoter activity

This article describes the use of three reporter enzymes used to study promoter activity in transgenic animals. Chloramphenicol acetyl transferase may be assayed by a nonchromatographic method that is rapid and sensitive. β-Galactosidase is measured by a photometric assay and luciferase is assayed by measuring the emission of light using a luminometer. The relative merits of each enzyme is discussed. Ths use of reporter enzymes provides a rapid and sensitive method for analysis of transgene expression.     

Stage-specific Monoclonal Antibodies to the Potato Cyst Nematode Globodera pallida Stone (Behrens)

Using standard hybridoma technology and hierarchical screening, monoclonal antibodies (MAbs) were obtained with specific reactivity against two developmental stages of Globodera pallida. The procedure was based on enzyme-linked immunosorbent assay (ELISA) with homogenates prepared from second-stage juveniles, young adult females, and potato roots. Hybridomas were formed by fusing myelomas with splenocytes derived from mice immunized with either infective juveniles or females of G. pallida. About 600 hybridoma lines were screened from the fusion involving the mouse immunized with juveniles. Two MAbs (LJMAbl &2) were identified with high reactivity toward second-stage juveniles but no reactivity with either potato roots or females of G. pallida. A total of 630 cell lines was screened from the corresponding fusion involving the spleen of a mouse receiving immunogens from adult female nematodes. One MAb (LFMAbl) was obtained with the required specificity against only adult female G. pallida. This work extends the application of monoclonal antibodies in nematology from valuable probes for research and species identification to recognition of developmental stages. These specific MAbs have potential value in plant breeding programs for screening for resistant lines unable to support nematode development.     

Combined effects of sulfites, temperature, and agitation time on production of glycerol in grape juice by Saccharomyces cerevisiae.

Analysis of variance was used to evaluate the simultaneous effects of strain, incubation temperature (15 to 25 degrees C), agitation time (0 to 24 h), and initial sulfite concentration (100 to 300 ppm) on glycerol production in grape juice by Saccharomyces cerevisiae. Fourteen strains were studied to determine their growth patterns in the presence of sulfites and ethanol. Baker's yeast strains were more sensitive to sulfite than wine strains, and little growth occurred at initial sulfite levels greater than 150 ppm. Sensitivity to sulfite increased with increasing levels of ethanol. Three strains exhibiting the best growth in the presence of sulfites and ethanol were selected for interaction studies. Fermentations were carried out until the solids content had decreased to less than 6 degrees Brix, which was the point that glycerol content became stable. For the three strains used, the greatest level of glycerol production was observed in the presence of 300 ppm of sulfite for most incubation temperatures and agitation times. There was significant interaction between the strain, incubation temperature, and agitation time parameters for glycerol synthesis, and a response surface method was used to predict the optimal conditions for glycerol production. Under static conditions, the highest level of glycerol production was observed at 20 degrees C, while incubation at 25 degrees C gave the best results when the cultures were agitated for 24 h. Response surface equations were used to predict that the optimum conditions for glycerol production by S. cerevisiae Y11 were a temperature of 22 degrees C, an initial sulfite concentration of 300 ppm, and no agitation, which yielded 0.68 g of glycerol per 100 ml.     

Methionine transamination—metabolic function and subcellular compartmentation

Enzymatic activities catalysing the inter-conversion of L-methionine and its oxy analogue 4-methylthio-2-oxobutyric acid (2,4-KMB) were detected in the liver, skeletal muscle and heart of the laboratory rat and of sheep. In both species the highest activity of methionine transamination was found in the liver and was located in the cytoplasm and mitochondria. We propose that physiological and nutritional role of the cytoplasmic methionine transamination is amination of 2,4 KMB and formation of L-methionine while in mitochondria the activity is responsible for disposal of excess methionine is oxidised through oxidative decarboxylation of 2,4 KMB.