ABSTRACT
Two components of the germ-line-specific P granules of the nematode Caenorhabditis elgans have been identified using polyclonal antibodies specific for each. Both components are putative germ-line RNA helicases (GLHs) that contain CCHC zinc fingers of the type found in the RNA-binding nucleocapsid proteins of retroviruses. The predicted GLH-1 protein has four CCHC fingers; GLH-2 has six. Both GLH proteins localize in the P granules at all stage of germ-line development. However, the two glh genes display different patterns of RNA and protein accumulation in the germ lines of hermaphrodites and males. Injection of antisense glh-1 or glh-2 RNA into wild-type worms causes some offspring to develop into sterile adults, suggesting that either or both genes are required for normal germ-line development. As these very similar glh genes physically map within several hundred kilobases of one another, it seems likely that they represent a fairly recent gene duplication event.
Subject(s)
Caenorhabditis elegans/physiology , Cytoplasmic Granules/enzymology , Gene Expression Regulation, Developmental , Multigene Family , RNA Nucleotidyltransferases/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans/enzymology , Chromosome Mapping , DNA Primers , Disorders of Sex Development , Embryo, Nonmammalian , Female , Male , Molecular Sequence Data , Polymerase Chain Reaction , RNA Helicases , RNA Nucleotidyltransferases/chemistry , RNA Nucleotidyltransferases/genetics , Retroviridae , Sequence Homology, Amino Acid , Spermatozoa/physiology , Zinc FingersABSTRACT
We have cloned a family of putative RNA helicases from the free-living nematode Caenorhabditis elegans. One of these, a cDNA that we call glh-1, most closely matches in sequence and expression the previously described germ-line helicases PL10 from mouse and vasa from Drosophila. The amino terminus of the predicted protein of glh-1 contains a set of glycine-rich repeats similar in location and sequence to those in the predicted vasa protein. However, unlike all other putative RNA helicases, glh-1 also contains four retroviral-type zinc fingers. The RNA expression pattern of this Caenorhabditis helicase correlates with the presence of germ-line tissue in the parasitic nematode Ascaris lumbricoides var. suum and with the presence of germ cells in wild type and several germ-line mutants of Caenorhabditis. In the germ-line mutants glp-4 and glp-1, additional larger species of glh-1 RNA exist, which correspond to different adenylylated forms of the glh-1 transcript; these may be specified by motifs in the 3' untranslated region of glh-1 that are similar to adenylylation control elements and nos response elements.
Subject(s)
Caenorhabditis/genetics , Genes, Helminth , RNA Nucleotidyltransferases/genetics , RNA-Binding Proteins/genetics , Zinc Fingers , Amino Acid Sequence , Animals , Chromosome Mapping , Consensus Sequence , Germ Cells/physiology , Molecular Sequence Data , Poly A/metabolism , RNA Helicases , RNA, Messenger/genetics , RNA-Binding Proteins/chemistry , Regulatory Sequences, Nucleic Acid , Repetitive Sequences, Nucleic Acid , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The nonchromosomal stripe 2 (NCS2) mutant of maize (Zea mays L.) has a DNA rearrangement in the mitochondrial genome that segregates with the abnormal growth phenotype. Yet, the NCS2 characteristic phenotype includes striped sectors of pale-green tissue on the leaves. This suggests a chloroplast abnormality. To characterize the chloroplasts present in the mutant sectors, we examined the chloroplast structure by electron microscopy, chloroplast function by radiolabeled carbon dioxide fixation and fluorescence induction kinetics, and thylakoid protein composition by polyacrylamide gel electrophoresis. The data from these analyses suggest abnormal or prematurely arrested chloroplast development. Deleterious effects of the NCS2 mutant mitochondria upon the cells of the leaf include structural and functional alterations in the both the bundle sheath and mesophyll chloroplasts.
ABSTRACT
Analysis of a series of clones containing deletions in the 5' noncoding sequence of a gene encoding an Mr 19,000 zein allowed identification of a region required for maximal transcription. Transcriptional activity was assayed in two heterologous plant systems. In one system, the Ti plasmid was used to introduce the modified zein genes into the sunflower genome. In the other system, electroporation was used to transform carrot protoplasts with plasmids containing the zein genes. For the electroporation experiments, the 5' noncoding sequences from the zein clones were linked to the protein coding sequence of chloramphenicol acetyl transferase. The results showed that an upstream sequence, delimited by nucleotides -337 and -125 with respect to the mRNA cap site, is required for maximal transcription of the gene. In contrast, very low levels of transcription were directed by constructs that contained 125 bp of 5' noncoding sequence that included the CAAT and TATA boxes, suggesting that the additional sequences (-337 to -125) further 5' exert a quantitative effect on transcription. Examination of the additional 5' sequences showed five regions that share homology with the SV40 enhancer core sequence.
