Protoplast production in Chondrus crispus gametophytes (gigartinales,rhodophyta)

Protoplasts were isolated from female gametophytes of Chondrus crispus (Stackh.) using commercial cellulase and various carrageenases prepared from marine bacteria. Depending on the nature of the donor tissue (apices or whole thallus, wild or cultivated strains), yields ranged from 1.0–8.5×10⁸ protoplasts per gram of fresh tissue. Preincubating the tissue with a potassium chelator, Kryptofix 222, enhanced protoplast yields by 30–50 %. Based on staining with fluorescein diacetate most protoplasts were viable. A few protoplasts regenerated a cell wall and divided.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

Isolation and chromosomal localization of the human glutathione peroxidase gene

We have isolated cDNA clones for the gene, termed GPX1, encoding the major human selenoprotein, glutathione peroxidase. Sequence analysis confirmed previous findings that the unusual amino acid seleno-cysteine is encoded by the opal terminator codon UGA. Southern blot analysis of human genomic DNA with the GPX1 cDNA showed that restriction endonucleases without sites in the probe sequence produced three hybridizing bands at standard stringency, diminishing to one strongly and one weakly hybridizing band at high stringency. In situ hybridization localized the human GPX1 gene to a single site on chromosome 3, at region 3q11-13.1. Thus, three genomic sites bear sequence homology to the GPX1 cDNA, and the one most homologous maps to 3q11-13.1.     

Cloning of cDNAs for human phosphoribosylpyrophosphate synthetases 1 and 2 and X chromosome localization of PRPS1 and PRPS2 genes

Cloned cDNAs representing the entire, homologous (80%) translated sequences of human phosphoribosylpyrophosphate synthetase (PRS) 1 and PRS 2 cDNAs were utilized as probes to localize the corresponding human PRPS1 and PRPS2 genes, previously reported to be X chromosome linked. PRPS1 and PRPS2 loci mapped to the intervals Xq22-q24 and Xp22.2-p22.3, respectively, using a combination of in situ chromosomal hybridization and human x rodent somatic cell panel genomic DNA hybridization analyses. A PRPS1-related gene or pseudogene (PRPS1L2) was also identified using in situ chromosomal hybridization at 9q33-q34. Human HPRT and PRPS1 loci are not closely linked. Despite marked cDNA and deduced amino acid sequence homology, human PRS 1 and PRS 2 isoforms are encoded by genes widely separated on the X chromosome.     

Influence of temperature and nitrogen concentration on photosynthesis ofDunaliella viridis Teodoresco

The photosynthetic behaviour ofDunaliella viridis has been studied under a combination of three variables: irradiance (0–900 mol m^–2 s^–1), temperature (15, 23, 31, 38, 42 °C) and nitrogen concentration (0.05, 0.5, 1.5, 5, 10 mM NO ₃ ^- ) at a salinity of 2 M NaCl.The highest rates of photosynthesis have been found at 31 °C and a nitrate concentration of 10 mM. There exists a synergistic effect between temperature and nitrogen availability on the photosynthesis ofD. viridis; under nitrogen deficiency oxygen evolution is low, even null at high temperature. The interaction between these two variables of control occurs in a multiplicative way. There is also a general increase in photosynthetic pigments following the increase in nitrogen concentration in the culture medium. The normalization of net photosynthesis data in relation to chlorophylla shows that nitrogen concentration makes an indirect control of the photosynthetic rate ofD. viridis through the variation of pigment concentration.     

Adipose-tissue-specific increase in glyceraldehyde-3-phosphate dehydrogenase activity and mRNA amounts in suckling pre-obese Zucker rats. Effect of weaning.

The regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was studied during the onset of obesity in the genetically obese (fa/fa) rat by determination of GAPDH activity and hybridizable mRNA amounts in adipose tissue and liver from suckling and weanling rats. GADPH activity remained low throughout the suckling period, and a burst of activity occurred after weaning in both lean and obese pups. As early as 7 days of age, adipose tissue from pre-obese rats displayed a significant increase in enzyme activity, whereas no difference could be detected in the liver. In both suckling (16 days of age) and weanling (30 days of age) obese rats a proportionate increase in GAPDH activity and mRNA amounts was observed in adipose tissue, but not in liver. It is concluded that the obese genotype influences GAPDH gene expression at a pretranslational level and in a tissue-specific manner. This phenomenon could partly contribute to the hyperactive fat accretion in the obese rat, since glycolysis is the major metabolic pathway for lipogenic substrates in adipose tissue.     

A group of type I keratin genes on human chromosome 17: characterization and expression.

The human type I keratins K16 and K14 are coexpressed in a number of epithelial tissues, including esophagus, tongue, and hair follicles. We determined that two genes encoding K16 and three genes encoding K14 were clustered in two distinct segments of chromosome 17. The genes within each cluster were tightly linked, and large parts of the genome containing these genes have been recently duplicated. The sequences of the two K16 genes showed striking homology not only within the coding sequences, but also within the intron positions and sequences and extending at least 400 base pairs 5' upstream and 850 base pairs 3' downstream from these genes. Despite the strong homologies between these two genes, only one of the genes encoded a protein which assembled into keratin filaments when introduced into simple epithelial cells. While there were no obvious abnormalities in the sequence of the other gene, its promoter seemed to be significantly weaker, and even a hybrid gene with the other gene's promoter gave rise to a much reduced mRNA level after gene transfection. To demonstrate that the functional K16 gene that we identified was in fact responsible for the K16 expressed in human tissues, we made a polyclonal antiserum which recognized our functional K16 gene product in both denatured and filamentous form and which was specific for bona fide human K16.     

The effect of beta-estradiol, progesterone and testosterone on the production of human leukocyte derived interferons

Sex hormones including estrogens, progesterone and testosterones are known to have adverse effects on the immune system and particularly on the proliferative response. Since cytokine production is known to be dissociable from the proliferation of lymphocytes and since other steroid hormones profoundly affect cytokine production, we felt it would be important to know the effect of sex steroids on the production of interferons (IFN), particularly since the latter are known to be key substances in the immune response. We have shown estradiol can slightly reduce gamma IFN yields with certain inducers (Con A, SEA) but only in pharmacologic concentrations. Similarly, progesterone had a modest effect in the same concentrations but only when Con A was the inducer. Testosterone did not effect IFN titers at any concentration. None of the sex steroids affected alpha IFN production and none of them influenced the bioactivity of either IFN species. In all cases these hormones diminished proliferative responses as has been previously noted.     

Interrenal activity in the female lizard Lacerta vivipara J.: in vitro response to ACTH 1-39 and to [Sar1, Val5] angiotensin II (ANG II)

A perifusion system technique was developed in order to determine in vitro the respective roles of ACTH and ANG II in the regulation of adrenal steroidogenesis in the lizard Lacerta vivipara. Synthetic human ACTH 1-39, administered as 20-min pulses, stimulated corticosterone (B) and aldosterone (A) release in a dose-dependent manner. The increase in corticosterone output was higher than that in aldosterone output, leading to an enhancement of the B/A ratio. Iterative stimulations with 1 nM ACTH (20-min pulses every 120 min) led to reproducible increases in corticosterone and aldosterone release. Prolonged stimulation with 1 nM ACTH (up to 240 min) caused a sustained increase in corticosteroid release, suggesting that, in the lizard, ACTH does not induce any desensitization phenomenon. The angiotensin II analogue [Sar1, Val5] ANG II also stimulated corticosterone and aldosterone release in a dose-dependent manner; the stimulatory effects of ANG II on both steroids were very similar. These results indicate that, in lizards, ACTH plays a major role in the regulation of adrenal steroidogenesis. Since ANG II stimulates the production of gluco- and mineralocorticoids, our data raise the question of the existence of two cell types synthesizing corticosterone and aldosterone, respectively, in reptiles.