Internal ribosome entry sites (IRESs): reality and use

IRESs are known to recruit ribosomes directly, without a previous scanning of untranslated region of mRNA by the ribosomes. IRESs have been found in a number of viral and cellular mRNAs. Experimentally, IRESs are commonly used to direct the expression of the second cistrons of bicistronic mRNAs. The mechanism of action of IRESs is not fully understood and a certain number of laboratories were not successful in using them in a reliable manner. Three observations done in our laboratory suggested that IRESs might not work as functionally as it was generally believed. Stem loops added before IRESs inhibited mRNA translation. When added into bicistronic mRNAs, IRESs initiated translation of the second cistrons efficiently only when the intercistronic region contained about 80 nucleotides, and they did not work any more effectively with intercistronic regions containing at least 300–400 nucleotides. Conversely, IRESs inserted at any position into the coding region of a cistron interrupted its translation and initiated translation of the following cistron. The first two data are hardly compatible with the idea that IRESs are able to recruit ribosomes without using the classical scanning mechanism. IRESs are highly structured and cannot be scanned by the 40S ribosomal subunit. We suggest that IRESs are shortcircuited and are essentially potent stimulators favoring translation in particular physiological situations.     

Hybrid origin of Japanese mice "Mus musculus molossinus": evidence from restriction analysis of mitochondrial DNA

The Japanese mouse, Mus musculus molossinus, has long been considered an independent subspecies of the house mouse. A survey of restriction- site haplotypes of mitochondrial DNA (mtDNA) showed that Japanese mice have two main maternal lineages. The most common haplotype is closely related to the mtDNA of the European subspecies M. m. musculus. The other common haplotype and two minor ones are closely related to each other and to the mtDNA of an Asiatic subspecies, M. m. castaneus. Two other rare variants are probably the result of recent contamination by European M. m. domesticus. The musculus type of mtDNA is found in the southern two-thirds of Japan, whereas the common castaneus type is found in the northern third and the minor variants are found sporadically throughout Japan. The castaneus mtDNA lineage had a few minor variants, whereas the musculus lineage was completely monomorphic. By contrast, the native population of M. m. castaneus and the Chinese and Korean musculus populations were highly polymorphic. These results suggest that M. m. molossinus is a hybrid between ancestral colonies, possibly very small, of M. m. musculus and M. m. castaneus, rather than an independent subspecies.      

Interferon effects on microfilament organization cellular fibronectin distribution, and cell motility in human fibroblasts

We have shown previously (Pfeffer et al., 1979, Exp. Cell Res. 121:111-120) that treatment of human fibroblasts, planted at a density of 2x10(3) cells/cm(2), with purified human fibroblasts interferon (640 U/ml) for 3 d at 37 degrees C decreases the overall rate of cell proliferation to 35-40 percent of the control value. In the present experiments we have characterized the phenotype of interferon-inhibited fibroblasts. The mean volume of trypsinized, interferon-treated cells was increased 31 percent abover that of control cells. The interferon-treated population was much more heterogeneous than the control population with respect to volume, and there was a considerable overlap in the volume distributions of the two populations. The cell surface area was, on the average, increased 65 percent after interferon treatment. More than 80 percent of the treated cells had enlarged nuclei, many of which were lobed, and the fraction of binucleated cells was increased fivefold. After interferon treatment, over 40 percent of the cells showed large actin-containing fibers in the form of multiple parallel arrays. Fewer than 5 percent of the control cells contained such large actin fibers. The number of actin fibers of all sizes was tripled in the treated fibroblasts on a per cell basis and, calculated per unit surface area of the cells, the number was increased 82 percent. In contrast, 10-nm filaments and microtubules did not appear to be increased in number per unit surface area of the cells. The increases per cell in the abundance of these structures were directly related to increased cell size. After interferon treatment, fibronection was distributed in arrays of long filaments covering most portions of the cell surface. Interferon treatment markedly decreased the rate of cell locomotion as well as membrane ruffling and saltatory movements of intracellular granules.     

Isolation and characterization of casein mRNAs from lactating ewe mammary glands.

RNA from bound polysomers of lactating ewe's mammary gland directs the synthesis of the three major milk proteins (alphas, beta and kappa-caseins) in a cell-free system derived from rabbit reticuyocytes. The "in vitro" product was identified by immunoprecipitation with specific antibodies and by electrophoresis in SDS polyacrylamide gel. Each of these messengers was purified from 20 to 25 fold from total membrane-bound polysomal RNA using poly U-Sepharose chromatography. This purified fraction assayed in a reticulocyte cell-free system is able to direct also the synthesis of 2 minor secretory proteins (beta-lactoglobulin and alpha-lactalbumin). The messenger RNAs purified by hybridization to poly U-Sepharose have a sedimentation coefficient of about 12 S and an apparent molecular weight of approximatively 3.5 s 10-5 daltons was observed by polyacrylamide gel electrophoresis under denaturing contitions. This value which correspond to about 900 nucleotides is significantly greater than the number expected for coding milk proteins.     

A 17.6 kbp region located upstream of the rabbit WAP gene directs high level expression of a functional human protein variant in transgenic mouse milk.

Mitochondrial protein import involves the recognition of preproteins by receptors and their subsequent translocation across the outer membrane. In Neurospora crassa, the two import receptors, MOM19 and MOM72, were found in a complex with the general insertion protein, GIP (formed by MOM7, MOM8, MOM30 and MOM38) and MOM22. We isolated a complex out of S. cerevisiae mitochondria consisting of MOM38/ISP42, the receptor MOM72, and five new yeast proteins, the putative equivalents of N. crassa MOM7, MOM8, MOM19, MOM22 and MOM30. A receptor complex isolated out of yeast cells transformed with N. crassa MOM19 contained the N. crassa master receptor in addition to the yeast proteins. This demonstrates that the yeast complex is functional, and provides strong evidence that we also have identified the yeast MOM19.     

Beta-glucan and pectin derivatives stimulate prolactin secretion from hypophysis in vitro

Previous work has shown that various plant extracts administered to animals stimulate milk protein synthesis through the secretion of prolactin. It has also been shown that beta-glucan and pectin are the active molecules capable of stimulating prolactin release in vivo after intravenous injections. In this work, it is shown that beta-glucan and several pectin derivatives are able to stimulate prolactin secretion from hypophysis fragments incubated for 2 hr in a synthetic medium.