Effects of plasmid presence on growth and enzyme activity of Escherichia coli DH5α

Summary The effects of recombinant DNA propagation and gene expression on the physiology of the host cell was investigated using a series of copy number mutant plasmids. The plasmids at copy numbers of 30, 57, 115 and 501 per chromosome equivalent encoded constitutive production of the enzyme -lactamase. Ribose phosphate isomerase activity was relatively unaffected by plasmid presence, and glucose-6-phosphate dehydrogenase, fructose 1,6-diphosphate aldolase and fructose 1,6-diphosphatase activities were lower in plasmid-containing cells than in the plasmid-free host strain. Increasing copy number resulted in increased depression of enzyme activity levels. The results indicate that plasmid presence mediates subtle changes in the net expression of host enzymes involved in carbon metabolism. Responses of Escherichia coli DH5 in Evans medium to these plasmids differed substantially from responses of E. coli HB101 in rich medium.Offprint requests to: J. E. Bailey     

Actions of extracellular matrix on Sertoli cell morphology and function

Sertoli cells were isolated and cultured in the absence or presence of extracellular matrix (ECM) to determine whether ECM may influence Sertoli cell function on a molecular level. As previously described, a morphological analysis of the cells indicated that ECM allows the expression of a columnar histotype and the formation of junctional complexes. The combined actions of ECM and hormones were found to have a profound effect in promoting the expression of a polarized Sertoli cell morphology. In our investigation of the effects of ECM on Sertoli cells, we used transferrin and androgen-binding protein (ABP) production as biochemical markers of Sertoli cell function. The presence of ECM was found to cause a 25% increase in the basal level of transferrin production; however, ECM had no effect on the basal level of ABP production by Sertoli cells. Regulatory agents such as follicle-stimulating hormone (FSH) and a combination of FSH, insulin, retinol, and testosterone stimulated the production of both transferrin and ABP. The ability of hormones to stimulate these Sertoli cell functions was not influenced by the presence of ECM. Similar results were obtained with 2-microns- or 50-microns-thick ECM and with a seminiferous tubule biomatrix preparation. ECM was found to increase the maintenance of long-term Sertoli cell cultures; however, the decline in Sertoli cell functional integrity, which occurs during cell culture, was not affected by the presence of ECM. An additional functional parameter examined was the radiolabeled proteins secreted by Sertoli cells. ECM did not promote the production or affect the electrophoretic profile of Sertoli cell-secreted proteins under basal or hormonally stimulated conditions. Combined results indicated that although ECM allowed the expression of a normal Sertoli cell histotype, ECM had no major effects on the Sertoli cell functions analyzed nor on the hormonal regulation of these functions. The inability of ECM to affect Sertoli cell function on a molecular level is discussed with regard to environmental as opposed to regulatory cellular interactions. Our observations imply that dramatic effects of ECM on cell morphology do not necessarily correlate to subsequent effects on cellular function.     

Methylation of ribosomal cistrons in diploid and tetraploid Odontophrynus americanus (Amphibia,Anura)

Odontophrynus americanus (Amphibia, Anura) genomic DNA from diploid and tetraploid specimens was treated with restriction enzymes sensitive to cytosine and adenine methylation (5 meC and 6 meA). In both diploids and tetraploids a high proportion of the total DNA was not cleaved by 5 meC-sensitive enzymes as observed on agarose gels stained with ethidium bromide. The DNAs were transferred to nitrocellulose filters and hybridized with cloned fragments containing sequences of Xenopus laevis 28S and 18S ribosomal DNA (rDNA). A high level of methylation of the ribosomal repeat units was revealed by 5 meC-sensitive enzymes in blood, liver, kidney and testis tissues. Adenine was methylated to a lesser degree and similarly in the rDNA from both germinative and somatic tissues. Comparison of the results obtained with DNA of diploids and tetraploids showed that methylation of ribosomal genes was increased in tetraploid genomes of adult frogs, but exact quantitative determinations could not be performed by this methodology. Cloning of the 28S region of the rDNA repeat unit was performed in the gtWESC vector. Restriction patterns obtained with methylation-sensitive enzymes using diploid and tetraploid derived clones confirmed the high level of methylation of the corresponding region of the ribosomal repeat unit in genomic DNAs. The implications of these results in the regulation of expression of the ribosomal genes in diploids and tetraploids are discussed.     

Nucleotide sequence of the Methylobacterium extorquens AM1 moxF and moxJ genes involved in methanol oxidation

The nucleotide sequence has been determined for two genes involved in methanol oxidation in the facultative methylotroph, Methylobacterium extorquens AM1. The two genes are moxF, encoding the 66-kDa subunit of the methanol dehydrogenase and moxJ, located immediately downstream from moxF, which encodes a 30-kDa protein with unknown function. This information completes the sequence of the 5.86-kb XhoI-SalI fragment containing the moxFJGI region in M. extorquens AM1, and the structure of this gene cluster is presented. Evidence is presented that moxJ is also present in Paracoccus denitrificans. The aa sequence of MoxJ has provided little information concerning its function, but it does appear to contain a signal sequence suggesting a periplasmic location.     

Developmental appearance of the Ca2+-binding proteins parvalbumin,calbindin D-28K,S-100 proteins and calmodulin during testicular development in the rat

Summary Calcium and intracellular Ca²⁺-binding proteins are possibly involved in hormone production and spermatogenesis in rat testis. Parvalbumin, calbindin D-28K, S-100 proteins and calmodulin were localized in the Leydig cells, which are sites of testosterone synthesis. Only the appearance of parvalbumin-immunoreactivity is closely correlated to testosterone production during development of the testes. Calbindin D-28K-immunoreactivity persisted in foetal-type Leydig cells and in adult-type Leydig cells at all stages of development. S-100-immunoreactivity was low during all foetal stages, absent between birth and puberty, and increased thereafter. Calmodulin staining is most prominent in the cytoplasm of developing spermatocytes and of maturing spermatids. All four proteins co-exist in the seminiferous tubules. The distinct localization and developmental appearance of these proteins suggests different regulatory roles in Leydig cell function and spermatogenesis.     

Sequence polymorphism of human complement factor H

Factor H is a major regulatory protein of the complement system. The complete cDNA coding sequence has been derived from overlapping clones, and a polymorphism at base 1277 has been characterized. In four clones there is a T at nucleotide 1277 and in two others there is a C. This T/C change represents a tyrosine/histidine polymorphism at position 384 in the derived amino acid sequence. Protein sequence studies on peptides generated by trypsin digestion of factor H, purified from pooled plasma from 12 donors, confirmed the presence of both tyrosine and histidine at this position. Tyrosine and histidine were observed in a ratio of 2 : 1, respectively, and therefore this polymorphism is likely to represent a sequence difference between the two most abundant charge variants, FH1 and FH2, of factor H.     

Ecology of microalgae in a high rate pond for piggery effluent purification in Singapore

Summary An integrated system for the collection, treatment and utilisation of piggery wastewater has been developed in Singapore which uses the cultivation of microalgae in high rate ponds to achieve reduction of BOD₅ and COD₅ of the effluent as well as producing single cell protein. A wide range of algal flora occurs in the ponds;Oocystis, Micratinium, Scenedesmus, Ankistrodesmus, Chlorella andOscillatoria spp were identified. Total algal counts, recorded from 1979 to 1981, ranged up to 10⁷ per ml of pond water. There were considerable variations in the algal population and in the predominating species. No discernible pattern was evident. Consequently pond operations were frequently disturbed by these fluctuations in population which in turn was attributed to the heterogeneous composition of the piggery waste, to variable weather conditions and to predation by larger organisms particularlyMoina. After passing through the ponds, the total suspended solids were removed by a novel dissolved air flotation method which gave a clear effluent showing an 87% reduction in BOD₅ value.

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Resumen En Singapur se ha desrrollado un sistema integrado para le recogida, tratamiento y utilización de aguas residuales de pocilgas. Este sistema usa el cultivo de microalgas en estanques, de caudal rápido afin de reducir las DBO₅ y DCO₅ del efluencte produciendo asimismo proteínas celulares. La flora de algas producida en estos estanques es amplia y variada, habiendose identificado:Oocystis, Micratinium, Scenesdemus, Ankistrodesmus, Chlorella y Oscillatoria. Los recuentos totales de algas tomados desde 1979 a 1981 llegaron a alcanzar 10;⁷ por ml de agua del estanque. Se observaron variaciones considerables tanto en la población total de algas como en las especies predominantes sin que se pudiese, determinar un patrón de variación característico. Estas fluctuaciones en la población, causantes de frecuentes alteraciones en el funcionamiento del estanque, se atribuyeron a la composición heterogenea de los resuduos, a las variaciones climáticas y la predación por otros organismos particularmenteMoina Después de su paso por los estanque los solidos suspendidos totales se eliminaron mediante un nuevo método de flotación con aire disuelto, obteniendose un efluente limpio con una reducción de la DBO₅ del 87%.

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Résumé Un système intégré pour la collecte, le traitement et l'utilisation d'eaux résiduaires de porcherie a été développé à Singapoure. Il utilise la culture de micro-algues tant pour la réduction accélérée en lagune de la DBO₅ et de la DCO de l'effluent que pour la production de protéines uni-cellulaires. On trouve une large gamme de flore algale dans les lagunes; des espèces d'-Oocystis, deMicratinium, deScenedesmus, Ankistrodesmus, deChlorella etd'Oscillatoria out été identifiées. Les comptages totaux d'algues, enregistrées de 1979 à 1981, ont donné jusqu' à 10⁷ cellules par ml d'eau de la lagune. On a observé des variations considérables de population algale tant quantitatives que qualitatives. On ne discernait pas de spectres évidents. En conséquence, les opérations lagunaires ont été fréquemment perturbées par ces fermentations en population, qui, à leur tour, ont été attribuées à la composition hétérogène de l'effluent de porcherie, aux conditions atmosphériques et climatiques variables et à la prédation par des organismes plus conséquents, plus particulièrement desMoina. Après passage par les lagunes, les solides totaux en suspension ont été enlevés par une méthode nouvelle de flottation à l'air dissous, qui a donné un effluent limpide, présentant une réduction de DBO₅ de 87%.