Structure and regulation of the sheep metallothionein-Ia gene

Screening of a sheep genomic lambda library with a sheep metallothionein-I cDNA clone resulted in the isolation of a 13,200-base-pair fragment containing a metallothionein gene which DNA sequence analysis identified as the gene encoding the cloned cDNA. The two introns occur at identical positions to those in other mammalian metallothioneins but are considerably larger. The first intron contains a DNA element that is present in a related but not identical form in many places in the sheep genome. Comparison of the promoter sequences of this gene (sMT-Ia) with the promoters of metallothionein genes from other species identified a number of conserved regions which may be important in the regulation of this gene by heavy metals, glucocorticoids and alpha-interferon. In sheep fibroblasts, the levels of sMT-Ia mRNA was found to be maximally elevated (95-fold) in the presence of zinc or cadmium and elevated 30-fold in the presence of copper. Dexamethasone had no effect upon mRNA levels. Thus this gene shows a pattern of regulation similar to the human MT-If gene, but distinct from the other human and mouse metallothionein genes so far reported.     

Inherited X-chromosome inverted tandem duplication in a male traced to a grandparental mitotic error.

A male infant was referred for cytogenetic evaluation because of dysmorphic features and developmental delay. In both lymphocytes and skin fibroblasts, a modal number of 46 chromosomes was obtained with an obvious elongation of the long arm of the X chromosome (Xq+). Studies of seven members in 3 generations of this family showed that the proband's mother, sister, and maternal grandmother were phenotypically normal carriers of this abnormal X chromosome. High resolution GTG- and RBG-banding defined the extra chromatin material as an inverted duplication of Xq21----Xq24. This was supported by an approximate twofold increase in alpha-galactosidase A activity, localized to Xq21----q24, observed in the proband's lymphocytes and fibroblasts. BrdU-incorporation studies of the mother's lymphocytes showed the abnormal X to be late replicating in all 100 cells studied and normal alpha-galactosidase A levels. Cytogenetic analysis of the maternal grandmother revealed cytogenetic mosaicism with one cell line containing the abnormal X (37%), and the other, a normal female karyotype (63%). This family is instructive since: (1) it represents only the second case of a dysmorphic male demonstrating a confirmed interstitial partial Xq duplication, and (2) the origin of this familial structural rearrangement has been traced to a grandparental mitotic error.     

Single turnover studies with oxy-cytochrome P-450cam

The catalytic step of bacterial cytochrome P-450cam, i.e., the step of the reaction cycle in which the product 5-exo-hydroxycamphor is formed and released by the enzyme, has been studied by stopped-flow spectrophotometry. Our approach has been to observe a single-turnover reaction between reduced putidaredoxin and oxygenated camphor-bound cytochrome P-450cam. Multiple turnovers are prevented by using the inhibitor metyrapone to trap the cytochrome after product release, which prevents binding of another camphor molecule. The time course of the reaction has been measured at several wavelengths and has been found to be biphasic. The relatively slow second phase of the reaction is the reduction of ferric, metyrapone-bound cytochrome P-450cam. The first phase coincides with the formation of product stoichiometrically with cytochrome P-450cam, as measured by gas chromatography. A detailed kinetic study of the first phase reveals a hyperbolic dependence of initial rate upon putidaredoxin concentration at a fixed, limiting concentration of cytochrome P-450cam. The Vmax is 53 microM per second per microM cytochrome, and the Km for putidaredoxin is 33 microM. The hyperbolic relationship between initial rate and putidaredoxin concentration supports a model in which the cytochrome rapidly binds putidaredoxin, then undergoes one or more slower intracomplex steps.     

Microfilaments appear in boar spermatozoa during capacitation in vitro

Boar spermatozoa were incubated in a capacitation medium and examined for the presence of filamentous actin by using the fluorescent probe NBD-phallacidin. F-actin was not observed in uncapacitated sperm, but developed in most regions of the cell during the capacitation period. Fluorescent staining was most intense in the flagellum. When fresh seminal plasma was added to capacitated sperm and the sperm was further incubated, F-actin was no longer observed. In view of previous experiments which indicated that plasma membrane proteins (PMPs), including a major integral PMP, move out of the sperm head into the flagellum during capacitation and that this movement is inhibited by the microfilament poison cytochalasin D (Peterson, Saxena, Saxena, and Russell: Biol. Reprod., in press, '86), we suggest that actin-PMP interactions play a major role in capacitating boar spermatozoa.     

Mineral licks as a sodium source for Isle Royale moose

Summary Natural mineral licks and their use by moose (Alces alces) on Isle Royale National Park, Michigan, were studied during 1982–85. The distribution of known licks suggested that they occurred in association with glacial debris, primarily in the western portions of the island. Moose utilized mineral springs extensively during the spring-summer period, and at least 5 licks were used year-round. During summer, a pronounced diel pattern of moose visitation was apparent, with peak use occurring between 0400–0800 h. Although daytime lick use declined by late June, morning and evening use continued to be relatively high throughout the study period. Peak lick use coincided with leaf-emergence in spring. Moose continued to utilize mineral licks despite the availability of ponds containing aquatic plants. Sodium appeared to be the element attracting moose to licks where they ingest copious amounts of water. Observed sodium ingestion rates (0.35 g/min) at licks indicate that licks provide a more concentrated source of sodium compared to aquatic plants (0.023 g/min). Based on the data presented, we reject the conclusions of earlier workers that aquatic plants constitute the only significant source of sodium for Isle Royale moose.     

Molecular cloning of the c2 locus of Zea mays, the gene coding for chalcone synthase

Summary The c2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable element En (Spm) as a gene tag. The Spm element present at the c2 locus in the autonomously mutating c2-m1 line was isolated using En1 element specific probes. Sequences flanking the element were identified as c2 locus specific and were used to clone the nonautonomous c2-m2 and wild-type alleles. The cloning and analysis of a cDNA complementary to the c2 locus provided evidence that this gene encodes the enzyme chalcone synthase.     

Changes in Insulin Binding to Developing Embryonic Chick Neural Retina Cells

Specific cell surface insulin binding to embryonic chick neural retina cells has been demonstrated in vivo. Kinetics of insulin binding as well as hormonal specificity were similar to those reported for other vertebrate cells and tissues, both neural and nonneural. When surface insulin binding to retinal cells was studied as a function of embryonic age, a developmental relationship was observed. Scatchard analysis revealed that the number of cell surface insulin receptors decreased approximately 75% between days 10 and 16 of embryonic development. Receptor affinities remained fairly constant for this period.     

Abrogation of cell surface expression of human class I transplantation antigens by an adenovirus protein in Xenopus laevis oocytes

Class I transplantation antigens form complexes with a virus protein encoded in the early region E3 of the adenovirus-2 genome. The interaction between this viral glycoprotein, E19, and nascent human class I antigens has been examined by microinjecting purified mRNA into Xenopus laevis oocytes. Both E19 and the two class I antigen subunits, the heavy chain and beta 2-microglobulin (beta 2M), were efficiently translated. The heavy chains did not become terminally glycosylated, as monitored by endoglycosidase H digestion, and were not expressed on the oocyte surface unless they were associated with beta 2M. The E19 protein did not become terminally glycosylated, and we failed to detect this viral protein on the surface of the oocytes. Co-translation of heavy chain and E19 mRNA demonstrated that the two proteins associate intracellularly. However, neither protein appeared to be transported to the trans-Golgi compartment. Similar observations were made in adenovirus-infected HeLa cells. Heavy chains bound to beta 2M became terminally glycosylated in oocytes in the presence of low concentrations of E19. At high concentrations of the viral protein, no carbohydrate modifications and no cell surface expression of class I antigens were apparent. Thus, beta 2M and E19 have opposite effects on the intracellular transport of the heavy chains. These data suggest that adenovirus-2 may impede the cell surface expression of class I antigens to escape immune surveillance.     

Fine specificity, structure, and proteolytic susceptibility of the human lymphocyte receptor for IgE

Properties of the Fc receptor for IgE (FC epsilon R) on cultured human B lymphoblastoid cells (RPMI 8866) were studied. Specificity for human IgE (hIgE) was demonstrated by inhibition studies with both Fc epsilon R+ intact cell and detergent-solubilized receptor preparations. No interaction of the FC epsilon R with other hIg classes or with rodent IgE was seen. In other studies, 3,3-dithiobis(sulfosuccinimidyl) propionate was used to cross-link hIgE to 125I surface-labeled 8866 cells. After detergent solubilization, the 125I receptor components were isolated by immunoprecipitation, and receptor peptides of 83 and 46 kilodalton kD were demonstrated by SDS-PAGE in the presence of reducing agents. Cross-linking performed after detergent solubilization gave identical results. Tryptic maps of the 83 and 46 kD polypeptides were identical with respect to surface-iodinated peptides; this indicates a structural homology between these components. The 83 kD component was more difficult to elute from IgE affinity columns, potentially because of an increased number of IgE binding sites per FC epsilon R molecule. Limited proteolysis studies of the purified FC epsilon R with papain and V8 protease from Staphylococcus aureus demonstrated that a 16 kD FC epsilon R fragment was rapidly produced. This component was also seen after papain treatment of intact cells, and it retained the ability to interact with anti-FC epsilon R antisera and, at least in the absence of detergent, with hIgE affinity columns. Potential relationships between the FC epsilon R and lymphokines that modulate the IgE response (IgE-binding factors) are discussed.