Nucleotide sequences of Caenorhabditis elegans core histone genes. Genes for different histone classes share common flanking sequence elements

We have determined the nucleotide sequence of core histone genes and flanking regions from two of approximately 11 different genomic histone clusters of the nematode Caenorhabditis elegans. Four histone genes from one cluster (H3, H4, H2B, H2A) and two histone genes from another (H4 and H2A) were analyzed. The predicted amino acid sequences of the two H4 and H2A proteins from the two clusters are identical, whereas the nucleotide sequences of the genes have diverged 9% (H2A) and 12% (H4). Flanking sequences, which are mostly not similar, were compared to identify putative regulatory elements. A conserved sequence of 34 base-pairs is present 19 to 42 nucleotides 3' of the termination codon of all the genes. Within the conserved sequence is a 16-base dyad sequence homologous to the one typically found at the 3' end of histone genes from higher eukaryotes. The C. elegans core histone genes are organized as divergently transcribed pairs of H3-H4 and H2A-H2B and contain 5' conserved sequence elements in the shared spacer regions. One of the sequence elements, 5' CTCCNCCTNCCCACCNCANA 3', is located immediately upstream from the canonical TATA homology of each gene. Another sequence element, 5' CTGCGGGGACACATNT 3', is present in the spacer of each heterotypic pair. These two 5' conserved sequences are not present in the promoter region of histone genes from other organisms, where 5' conserved sequences are usually different for each histone class. They are also not found in non-histone genes of C. elegans. These putative regulatory sequences of C. elegans core histone genes are similar to the regulatory elements of both higher and lower eukaryotes. The coding regions of the genes and the 3' regulatory sequences are similar to those of higher eukaryotes, whereas the presence of common 5' sequence elements upstream from genes of different histone classes is similar to histone promoter elements in yeast.     

Site-selected insertion of the transposon Tc1 into a Caenorhabditis elegans myosin light chain gene.

We used the polymerase chain reaction to detect insertions of the transposon Tc1 into mlc-2, one of two Caenorhabditis elegans regulatory myosin light chain genes. Our goals were to develop a general method to identify mutations in any sequenced gene and to establish the phenotype of mlc-2 loss-of-function mutants. The sensitivity of the polymerase chain reaction allowed us to identify nematode populations containing rare Tc1 insertions into mcl-2. mlc-2::Tc1 mutants were subsequently isolated from these populations by a sib selection procedure. We isolated three mutants with Tc1 insertions within the mlc-2 third exon and a fourth strain with Tc1 inserted in nearby noncoding DNA. To demonstrate the generality of our procedure, we isolated two additional mutants with Tc1 insertions within hlh-1, the C. elegans MyoD homolog. All of these mutants are essentially wild type when homozygous. Despite the fact that certain of these mutants have Tc1 inserted within exons of the target gene, these mutations may not be true null alleles. All three of the mlc-2 mutants contain mlc-2 mRNA in which all or part of Tc1 is spliced from the pre-mRNA, leaving small in-frame insertions or deletions in the mature message. There is a remarkable plasticity in the sites used to splice Tc1 from these mlc-2 pre-mRNAs; certain splice sites used in the mutants are very different from typical eukaryotic splice sites.     

A uniquely conserved regulatory signal is found around the translation initiation site in three different collagen genes

We have determined the nucleotide sequence of the 5' untranslated region and the sequence encoding the signal peptide for mRNAs of the chick alpha 1 type I and alpha 1 type III collagen. These sequences were obtained by synthesizing the corresponding cDNAs using as primers either a synthetic oligonucleotide to prime alpha 1 type I cDNA or a DNA fragment isolated from a genomic clone coding for alpha 1 type III collagen to prime the cognate cDNA. Both primers were selected so that the resulting cDNAs would be short and would contain sequence information for the 5' untranslated region and the signal peptide of the proteins. The nucleotide sequences of these cDNAs were compared with the corresponding sequence of alpha 2 type I collagen. In each mRNA the 5' untranslated segment is approximately 130 nucleotides and contains two or more AUG triplets preceding the AUG which serves as a translation initiation codon. A sequence of about 50 nucleotides surrounding the translation initiation codon is remarkably conserved in all three mRNAs, whereas the sequences preceding and following this segment diverge markedly. This homologous sequence contains an almost identical inverted repeat sequence which could form a stable stem-loop structure. The initiation codon and the AUG which precedes it are found at the same place within this symmetrical sequence and the distance between them is invariant. The rest of the conserved sequence shows a less perfect symmetry. This conserved sequence has not been found in other genes. Our data suggest that these three and perhaps other collagen genes contain an identical regulatory signal that may play a role in determining the level of expression of these genes by modulating translational efficiency.     

Functions of the Caenorhabditis elegans regulatory myosin light chain genes mlc-1 and mlc-2.

Caenorhabditis elegans contains two muscle regulatory myosin light chain genes, mlc-1 and mlc-2. To determine their in vivo roles, we identified deletions that eliminate each gene individually and both genes in combination. Functions of mlc-1 are redundant to those of mlc-2 in both body-wall and pharyngeal muscle. mlc-1(0) mutants are wild type, but mlc-1(0) mlc-2(0) double mutants arrest as incompletely elongated L1 larvae, having both pharyngeal and body-wall muscle defects. Transgenic copies of either mlc-1(+) or mlc-2(+) rescue all defects of mlc-1(0) mlc-2(0) double mutants. mlc-2 is redundant to mlc-1 in body-wall muscle, but mlc-2 performs a nearly essential role in the pharynx. Approximately 90% of mlc-2(0) hermaphrodites arrest as L1 larvae due to pharyngeal muscle defects. Lethality of mlc-2(0) mutants is sex specific, with mlc-2(0) males being essentially wild type. Four observations suggest that hermaphrodite-specific lethality of mlc-2(0) mutants results from insufficient expression of the X-linked mlc-1(+) gene in the pharynx. First, mlc-1(0) mlc-2(0) double mutants are fully penetrant L1 lethals in both hermaphrodites and males. Second, in situ localization of mlc mRNAs demonstrates that both mlc-1 and mlc-2 are expressed in the pharynx. Third, transgenic copies of either mlc-1(+) or mlc-2(+) rescue the pharyngeal defects of mlc-1(0) mlc-2(0) hermaphrodites. Fourth, a mutation of the dosage compensation gene sdc-3 suppresses hermaphrodite-specific lethality of mlc-2(0) mutants.     

Identification and Characterization of the Functional Elements within the Tobacco Etch Virus 5′ Leader Required for Cap-Independent Translation

Translation in plants is highly cap dependent, and the only plant mRNAs known to naturally lack a cap structure (m(7)GpppN) are viral in origin. The genomic RNA of tobacco etch virus (TEV), a potyvirus that belongs to the picornavirus superfamily, is a polyadenylated mRNA that is naturally uncapped and yet is a highly competitive mRNA during translation. The 143-nucleotide 5' leader is responsible for conferring cap-independent translation even on reporter mRNAs. We have carried out a deletion analysis of the TEV 5' leader to identify the elements responsible for its regulatory function and have identified two centrally located cap-independent regulatory elements (CIREs) that promote cap-independent translation. The introduction of a stable stem-loop structure upstream of each element demonstrated that CIRE-1 is less 5' end dependent in function than CIRE-2. In a dicistronic mRNA, the presence of the TEV 5' leader sequence in the intercistronic region increased expression of the second cistron, suggesting that the viral sequence can function in a 5'-distal position. Interestingly, the introduction of a stable stem-loop upstream of the TEV leader sequence or upstream of either CIRE in dicistronic constructs markedly increased their regulatory function. These data suggest that the TEV 5' leader contains two elements that together promote internal initiation but that the function of one element, in particular, is facilitated by proximity to the 5' end.     

Sequence Requirements for Myosin Gene Expression and Regulation in Caenorhabditis Elegans

Four Caenorhabditis elegans genes encode muscle-type specific myosin heavy chain isoforms: myo-1 and myo-2 are expressed in the pharyngeal muscles; unc-54 and myo-3 are expressed in body wall muscles. We have used transformation-rescue and lacZ fusion assays to determine sequence requirements for regulated myosin gene expression during development. Multiple tissue-specific activation elements are present for all four genes. For each of the four genes, sequences upstream of the coding region are tissue-specific promoters, as shown by their ability to drive expression of a reporter gene (lacZ) in the appropriate muscle type. Each gene contains at least one additional tissue-specific regulatory element, as defined by the ability to enhance expression of a heterologous promoter in the appropriate muscle type. In rescue experiments with unc-54, two further requirements apparently independent of tissue specificity were found: sequences within the 3' non-coding region are essential for activity while an intron near the 5' end augments expression levels. The general intron stimulation is apparently independent of intron sequence, indicating a mechanistic effect of splicing. To further characterize the myosin gene promoters and to examine the types of enhancer sequences in the genome, we have initiated a screen of C. elegans genomic DNA for fragments capable of enhancing the myo-2 promoter. The properties of enhancers recovered from this screen suggest that the promoter is limited to muscle cells in its ability to respond to enhancers.     

Multiple ribonuclease H-encoding genes in the Caenorhabditis elegans genome contrasts with the two typical ribonuclease H-encoding genes in the human genome

Database searches of the Caenorhabditis elegans and human genomic DNA sequences revealed genes encoding ribonuclease H1 (RNase H1) and RNase H2 in each genome. The human genome contains a single copy of each gene, whereas C. elegans has four genes encoding RNase H1-related proteins and one gene for RNase H2. By analyzing the mRNAs produced from the C. elegans genes, examining the amino acid sequence of the predicted protein, and expressing the proteins in Esherichia coli we have identified two active RNase H1-like proteins. One is similar to other eukaryotic RNases H1, whereas the second RNase H (rnh-1.1) is unique. The rnh-1.0 gene is transcribed as a dicistronic message with three dsRNA-binding domains; the mature mRNA is transspliced with SL2 splice leader and contains only one dsRNA-binding domain. Formation of RNase H1 is further regulated by differential cis-splicing events. A single rnh-2 gene, encoding a protein similar to several other eukaryotic RNase H2L's, also has been examined. The diversity and enzymatic properties of RNase H homologues are other examples of expansion of protein families in C. elegans. The presence of two RNases H1 in C. elegans suggests that two enzymes are required in this rather simple organism to perform the functions that are accomplished by a single enzyme in more complex organisms. Phylogenetic analysis indicates that the active C. elegans RNases H1 are distantly related to one another and that the C. elegans RNase H1 is more closely related to the human RNase H1. The database searches also suggest that RNase H domains of LTR-retrotransposons in C. elegans are quite unrelated to cellular RNases H1, but numerous RNase H domains of human endogenous retroviruses are more closely related to cellular RNases H.     

Binding specificity and mRNA targets of a C. elegans PUF protein, FBF-1

Sequence-specific RNA-protein interactions underlie regulation of many mRNAs. Here we analyze the RNA sequence specificity of Caenorhabditis elegans FBF-1, a founding member of the PUF protein family. Like other PUF proteins, FBF-1 binds to the 3' UTR of target mRNAs and decreases expression of those target genes. Here, we show that FBF-1 and its close relative, FBF-2, bind with similar affinity to multiple RNA sites. We use mutagenesis and in vivo selection experiments to identify nucleotides that are essential for FBF-1 binding. The binding elements comprise a "core" central region and flanking sequences. The core region is similar but distinct from the binding sites of other PUF proteins. We combine the identification of binding elements with informatics to predict new FBF-1 binding sites in a C. elegans 3' UTR database. These data identify a set of new candidate mRNA targets of FBF-1 and FBF-2.     

Operon structure and trans-splicing in the nematode Pristionchus pacificus

In the nematode Caenorhabditis elegans, up to 15% of the genes are organized in operons. Polycistronic precursor RNAs are processed by trans-splicing at the 5' ends of genes by adding a specific trans-spliced leader. Ten different spliced leaders are known in C. elegans that differ in sequence and abundance. The SL1 leader is most abundant and is spliced to the 5' ends of monocistronic genes and to upstream genes in operons. Trans-splicing is common among nematodes and was observed in the genera Panagrellus, Ascaris, Haemonchus, Anisakis, and Brugia. However, little is known about operons in nonrhabditid nematodes. Dolichorhabditis CEW1, another rhabditid nematode that is now called Oscheius CEW1, contains operons and SL2 trans-splicing. We have studied the presence of operons and trans-splicing in Pristionchus pacificus, a species of the Diplogastridae that has recently been developed as a satellite organism in evolutionary developmental biology. We provide evidence that P. pacificus contains operons and that downstream genes are trans-spliced to SL2. Surprisingly, the one operon analyzed so far in P. pacificus is not conserved in C. elegans, suggesting unexpected genomic plasticity.     

Molecular cloning and nucleotide sequences of the complementary DNAs to chicken skeletal muscle myosin two alkali light chain mRNAs.

We report here the molecular cloning and sequence analysis of DNAs complementary to mRNAs for myosin alkali light chain of chicken embryo and adult leg skeletal muscle. pSMA2-1 contained an 818 base-pair insert that includes the entire coding region and 5' and 3' untranslated regions of A2 mRNA. pSMA1-1 contained a 848 base-pair insert that included the 3' untranslated region and almost all of the coding region except for the N-terminal 13 amino acid residues of the A1 light chain. The 741 nucleotide sequences of A1 and A2 mRNAs corresponding to C-terminal 141 amino acid residues and 3' untranslated regions were identical. The 5' terminal nucleotide sequences corresponding to N-terminal 35 amino acid residues of A1 chain were quite different from the sequences corresponding to N-terminal 8 amino acid residues and of the 5' untranslated region of A2 mRNA. These findings are discussed in relation to the structures of the genes for A1 and A2 mRNA.     

The nonmuscle myosin regulatory light chain gene mlc-4 is required for cytokinesis, anterior-posterior polarity, and body morphology during Caenorhabditis elegans embryogenesis.

Using RNA-mediated genetic interference in a phenotypic screen, we identified a conserved nonmuscle myosin II regulatory light chain gene in Caenorhabditis elegans, which we name mlc-4. Maternally supplied mlc-4 function is required for cytokinesis during both meiosis and mitosis and for establishment of anterior-posterior (a-p) asymmetries after fertilization. Reducing the function of mlc-4 or nmy-2, a nonmuscle myosin II gene, also leads to a loss of polarized cytoplasmic flow in the C. elegans zygote, supporting models in which cytoplasmic flow may be required to establish a-p differences. Germline P granule localization at the time of cytoplasmic flow is also lost in these embryos, although P granules do become localized to the posterior pole after the first mitosis. This result suggests that a mechanism other than cytoplasmic flow or mlc-4/nmy-2 activity can generate some a-p asymmetries in the C. elegans zygote. By isolating a deletion allele, we show that removing zygotic mlc-4 function results in an elongation phenotype during embryogenesis. An mlc-4/green fluorescent protein transgene is expressed in lateral rows of hypodermal cells and these cells fail to properly change shape in mlc-4 mutant animals during elongation.     

Characterization of a Trypanosoma cruzi genomic fragment complementary to several species-specific mRNAs but different from the spliced leader sequence

We have isolated a clone of Trypanosoma cruzi genomic DNA, lambda 3b2-5, which contains sequences that are reiterated in the genome. Northern blot analysis showed that clone 3b2-5 hybridizes to 1,200-5,000 bases different mRNA species. The number of mRNAs species hybridized to clone 3b2-5 exceeds its coding capacity showing that this clone carries sequences that are common to several mRNAs species and conserved in the poly A(+) RNA. These sequences are not homologous to the T. cruzi spliced leader sequence, since clone 3b2-5 does not hybridize to a synthetic 20 nucleotide complementary to the spliced leader sequence. Clone 3b2-5 does not hybridize to DNA and RNA from several genera of Trypanosomatidae and other Trypanosoma species indicating that it carries T. cruzi species-specific sequences.