Clathrin light chain B: gene structure and neuron-specific splicing.

The clathrin light chains are components of clathrin coated vesicles, structural constituents involved in endocytosis and membrane recycling. The clathrin light chain B (LCB) gene encodes two isoforms, termed LCB2 and LCB3, via an alternative RNA splicing mechanism. We have determined the structure of the rat clathrin light chain B gene. The gene consists of six exons that extend over 11.9 kb. The first four exons and the last exon are common to the LCB2 and LCB3 isoforms. The fifth exon, termed EN, is included in the mRNA in brain, giving rise to the brain specific form LCB2 but is excluded in other tissues, generating the LCB3 isoform. Primary rat neuronal cell cultures express predominantly the brain specific LCB2 isoform, whereas primary rat cultures of glia express only the LCB3 isoform, suggesting that expression of the brain-specific LCB2 form is limited to neurons. Further evidence for neuronal localization of the LCB2 form is provided using a teratocarcinoma cell line, P19, which can be induced by retinoic acid to express a neuronal phenotype, concomitant with the induction of the LCB2 form. In order to determine the sequences involved in alternative splice site selection, we constructed a minigene containing the alternative spliced exon EN and its flanking intron and exon sequences. This minigene reflects the splicing pattern of the endogenous gene upon transfection in HeLa cell and primary neuronal cell cultures, indicating that this region of the LCB gene contains all the necessary information for neuron-specific splicing.     

Cloning of the recA gene from a free-living leptospire and distribution of RecA-like protein among spirochetes

A recombinant plasmid carrying the recA gene of Leptospira biflexa serovar patoc was isolated from a cosmid library of genomic DNA by complementation of an Escherichia coli recA mutation. The cloned serovar patoc recA gene efficiently restored resistance to UV radiation and methyl methanesulfonate. Recombination proficiency was also restored, as measured by the formation of Lac+ recombinants from duplicated mutant lacZ genes. Additionally, the cloned recA gene increased the spontaneous and mitomycin C-induced production of lambda phage in lysogens of an E. coli recA mutant. The product of the cloned recA gene was identified in maxicells as a polypeptide with an Mr of 43,000. Antibodies prepared against the E. coli RecA protein cross-reacted with the serovar patoc RecA protein, indicating structural conservation. Southern hybridization data showed that the serovar patoc recA gene has diverged from the recA gene of L. interrogans, Leptonema illini, and E. coli. With the exception of the RecA protein of L. interrogans serovar hardjo, the RecA protein of the Leptospira serovars and L. illini were synthesized at elevated levels following treatment of cells with nalidixic acid. The level of detectable RecA correlated with previous studies demonstrating that free-living cells of L. biflexa serovars and L. illini were considerably more resistant to DNA-damaging agents than were those of parasitic L. interrogans serovars. RecA protein was not detected in cells of virulent Treponema pallidum or Borrelia burgdorferi.     

Thylakoid differentiation in endocyanelles

Cyanophora paradoxa Korshikov synchronized autotrophically in a light-dark regime of 14 h light and 10 h dark divides in the last two hours of the dark period. The division rate of the free-living blue-green alga, Synechococcus leopoliensis Raciborski, at identical culture conditions (24°C; 32 W m⁻²) is only slightly lowered in the light period. The comparison of thylakoid differentiation in the endocyanelles of Cyanophora paradoxa and in Synechococcus leopoliensis during the light-dark regime yields (1) the same ensemble of pigment-protein complexes in both organisms, (2) comparable syntheses of chlorophyll and phycobilins of Cyanophora paradoxa grown under 32 W m⁻² and of Synechococcus leopoliensis grown under light intensities below 9.2 W m⁻², and (3) identical photosynthetic oxygen evolution during the light period of the light-dark regime with minima at the beginning, in the middle (6th–7th h), and at the end of the light period. In both organisms this stage-specific oxygen evolution is inhibited by treatment with chloroamphenicol. Cycloheximide, however, causes no significant alterations. Results are discussed in view of the endosymbiotic theory.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Effects of magnetic fields on mammary tumor development induced by 7, 12-dimethylbenz(a)anthracene in rats

A series of epidemiological studies have indicated associations between exposure to magnetic fields (MFs) and a variety of cancers, including breast cancer. In order to test the possibility that MF acts as a cancer promoter or copromoter, four separate experiments have been conducted in rats in which the effects of chronic exposure to MFs on the development of mammary tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA) were determined. Female rats were exposed in magnetic coils for 91 days (24 h/day) to either alternating current (AC; 50 Hz)-MF or direct current (DC)-MF. Magnetic flux density of the DC-MF was 15 mT. Two AC-MF exposures used a homogeneous field with a flux density of 30 mT (rms); one used a gradient field with flux density ranging from 0.3–1 μT. DMBA (5 mg) was administered orally at the onset of MF exposure and was repeated thrice at intervals of 1 week. In each experiment, 18–36 animals were exposed in 6 magnetic coils. The same number of rats were used as sham-exposed control. These control animals were treated with DMBA and were placed in dummy coils in the same room as the MF-exposed rats. Furthermore, groups of age-matched rats (reference controls) were treated with DMBA but housed in another room to exclude any MF exposure due to the magnetic stray field from the MF produced by coils. At the end of the exposure or sham-exposure period, tumor number and weight or size of tumors were determined at necropsy. Results were as follows: In sham-exposed animals or reference controls, the tumor incidence varied between 50 and 78% in the 4 experiments. The average number of mammary tumors per tumor-bearing animal varied between 1.6 and 2.9. In none of the experiments did MFs significantly alter tumor incidence, but in one of the experiments with AC-MF exposure at 30 mT, the number of tumors per tumor-bearing animal was significantly increased. Furthermore, exposure to a DC-MF at 15 mT significantly enhanced the tumor weight. Exposure to a gradient AC-MF at 0.3–1 μT exerted no significant effects. These experiments seem to indicate that MFs at high flux densities may act as a promoter or copromoter of breast cancer. However, this interpretation must be considered only a tentative conclusion because of the limitations of this study, particularly the small sample size used for MF exposure and the lack of repetition of data. © 1993 Wiley-Liss. Inc.     

Valproic Acid Causes Proteasomal Degradation of DICER and Influences miRNA Expression

Valproic acid (VPA) is a commonly used drug to treat epilepsy and bipolar disorders. Known properties of VPA are inhibitions of histone deacetylases and activation of extracellular signal regulated kinases (ERK), which cannot fully explain VPA’s clinical features. We found that VPA induces the proteasomal degradation of DICER, a key protein in the generation of micro RNAs. Unexpectedly, the concentration of several micro RNAs increases after VPA treatment, which is caused by the upregulation of their hosting genes prior to DICER degradation. The data suggest that a loss of DICER protein and changes in micro RNA concentration contributes to the clinical properties of VPA. VPA can be used experimentally to down regulate DICER protein levels, which likely reflects a natural regulation of DICER.