Repetitive sequences of the sea urchin genome: Distribution of members of specific repetitive families

Three repetitive sequence families from the sea urchin genome were studied, each defined by homology with a specific cloned probe one to a few hundred nucleotides long. Recombinant λ-sea urchin DNA libraries were screened with these probes, and individual recombinants were selected that include genomic members of these families. Restriction mapping, gel blot, and kinetic analyses were carried out to determine the organization of each repeat family. Sequence elements belonging to the first of the three repeat families were found to be embedded in longer repeat sequences. These repeat sequences frequently occur in small clusters. Members of the second repeat family are also found in a long repetitive sequence environment, but these repeats usually occur singly in any given region of the DNA. The sequences of the third repeat are only 200 to 300 nucleotides long, and are generally terminated by single copy DNA, though a few examples were found associated with other repeats. These three repeat sequence families constitute sets of homologous sequence elements that relate distant regions of the DNA.     

Interacting role of thyroxine and growth hormone in the hepatic synthesis of alpha 2u-globulin and its messenger RNA

Hypophysectomy completely abolishes and thyroidectomy results in a 90% reduction in the hepatic content of alpha 2u-globulin and its mRNA in the male rat. Thyroid hormone is also known to be required for the synthesis and secretion of pituitary growth hormone. In the hypothyroid rat either thyroxine or growth hormone was found to increase the activity and number of sequences of the mRNA for alpha 2u-globulin (measured by translational assay and hybridizational analysis with a cloned cDNA probe) to the euthyroid level. Treatment of hypophysectomized rats with a hormone combination containing growth hormone but not thyroxine increased the hepatic level of the mRNA for alpha 2u-globulin to that of normal animals. From these results we conclude that thyroxine indirectly influences the hepatic concentration of the mRNA for alpha 2u-globulin through its effect on pituitary growth hormone. Although administration of growth hormone to hypothyroid animals raised the hepatic concentration of alpha 2u-globulin mRNA to the euthyroid level, synthesis of alpha 2u-globulin remained low (50% of the normal). Complete recovery of alpha 2u-globulin synthesis required thyroxine. Therefore, in addition to an indirect effect on the hepatic level of alpha 2u-globulin mRNA, thyroxine also directly influences the synthesis of this protein. This direct effect of thyroxine on alpha 2u-globulin synthesis seems to be exerted at a step distal to the formation of mature mRNA.     

A simple quantitative method for the estimation of free ecto-sulfhydryl groups of spermatozoa

A simple rapid quantitative method has been developed for the estimation of sperm ecto-SH groups on the basis of their high affinity binding to the mercurial: [203Hg]p-chloromercuriphenylsulfonic acid (PCMPS) used as a surface probe. The thiol reagent did not penetrate the sperm plasma membrane, as evidenced by the extremely rapid time course of the binding reaction and undetectable uptake of [203Hg]PCMPS by intact goat spermatozoa. The binding reaction was not due to contaminating broken or damaged cells, if any. The method consists of incubating of highly motile goat spermatozoa with PCMPS in a modified Ringer solution at 37 degrees C for 5 min, agglutination of the labelled cells with polyethyleneimine (100 micrograms/ml) and filtration and washing of the cell suspension through Whatman No. 1 filter discs under mild vacuum. The binding interaction is proportional to cell concentration, specific and saturable at 50 microM PCMPS. The method is capable of estimating free ecto-SH as low as 25 pmoles. Spermatozoa possess 286 +/- 61 pmoles of free ecto-SH groups/10(6) cells. Scatchard analysis showed the presence in goat spermatozoa of multiple classes of ecto-SH groups differing in their affinity for PCMPS.     

A simulation approach for power calculation in large cohort studies based on multistate models

Realistic power calculations for large cohort studies and nested case control studies are essential for successfully answering important and complex research questions in epidemiology and clinical medicine. For this, we provide a methodical framework for general realistic power calculations via simulations that we put into practice by means of an R‐based template. We consider staggered recruitment and individual hazard rates, competing risks, interaction effects, and the misclassification of covariates. The study cohort is assembled with respect to given age‐, gender‐, and community distributions. Nested case‐control analyses with a varying number of controls enable comparisons of power with a full cohort analysis. Time‐to‐event generation under competing risks, including delayed study‐entry times, is realized on the basis of a six‐state Markov model. Incidence rates, prevalence of risk factors and prefixed hazard ratios allow for the assignment of age‐dependent transition rates given in the form of Cox models. These provide the basis for a central simulation‐algorithm, which is used for the generation of sample paths of the underlying time‐inhomogeneous Markov processes. With the inclusion of frailty terms into the Cox models the Markov property is specifically biased. An “individual Markov process given frailty” creates some unobserved heterogeneity between individuals. Different left‐truncation‐ and right‐censoring patterns call for the use of Cox models for data analysis. p‐values are recorded over repeated simulation runs to allow for the desired power calculations. For illustration, we consider scenarios with a “testing” character as well as realistic scenarios. This enables the validation of a correct implementation of theoretical concepts and concrete sample size recommendations against an actual epidemiological background, here given with possible substudy designs within the German National Cohort.