Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

Characterization of cloned ribosomal DNA from Drosophila hydei.

The structure of ribosomal genes from the fly Drosophila hydei has been analyzed. EcoRI fragments, cloned in a plasmid vector, were mapped by restriction enzyme analysis. The lengths of the regions coding for 18S and 28S rRNA were defined by R-loop formation. From these data a physical map of the rRNA genes was constructed. There are two major types of rDNA units in D. hydei, one having a size of 11 kb and the other a size of 17 kb. The 17 kb unit results from an intervening sequence (ivs) of 6.0 kb, interrupting the beta-28S rRNA coding region. Some homology between th D. hydei ivs and D. melanogaster type 1 ivs has been described previously (1). However, the restriction sites within these ivs show considerable divergence. Whereas D. hydei rDNA D. melanogaster rDNA, the nontranscribed spacer has little, if any, sequence homology. Despite difference in sequence, D. hydei and D. melanogaster spacers show structural similarities in that both contain repeated sequence elements of similar size and location.     

Molecular evolutionary analysis of a histone gene repeating unit from Drosophila simulans

A repeating unit of the histone gene cluster from Drosophila simulans containing the H1, H2A, H2B and H4 genes (the H3 gene region has already been analyzed) was cloned and analyzed. A nucleotide sequence of about 4.6 kbp was determined to study the nucleotide divergence and molecular evolution of the histone gene cluster. Comparison of the structure and nucleotide sequence with those of Drosophila melanogaster showed that the four histone genes were located at identical positions and in the same directions. The proportion of different nucleotide sites was 6.3% in total. The amino acid sequence of H1 was divergent, with a 5.1% difference. However, no amino acid change has been observed for the other three histone proteins. Analysis of the GC contents and the base substitution patterns in the two lineages, D. melanogaster and D. simulans, with a common ancestor showed the following. 1) A strong negative correlation was found between the GC content and the nucleotide divergence in the whole repeating unit. 2) The mode of molecular evolution previously found for the H3 gene was also observed for the whole repeating unit of histone genes; the nucleotide substitutions were stationary in the 3' and spacer regions, and there was a directional change of the codon usage to the AT-rich codons. 3) No distinct difference in the mode or pattern of molecular evolution was detected for the histone gene repeating unit in the D. melanogaster and D. simulans lineages. These results suggest that selectional pressure for the coding regions of histones, which eliminate A and T, is less effective in the D. melanogaster and D. simulans lineages than in the other GC-rich species.     

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.     

The Evolution of Duplicate Glyceraldehyde-3-Phosphate Dehydrogenase Genes in Drosophila

In Drosophila melanogaster there are two genes which encode the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Gapdh-43E and Gapdh-13F. We have shown that Gapdh-43E codes for the GAPDH subunit with an apparently larger molecular weight while Gapdh-13F encodes the GAPDH subunit having an apparently smaller molecular weight. Immunoblots of sodium dodecyl sulfate gels were used to survey species from throughout the genus and results indicated that two classes of GAPDH subunits are present only in Drosophila species of the melanogaster and takahashi subgroups of the melanogaster group. Only the smaller subunit is found in species of the obscura group while all other species have only a large subunit. Drosophila hydei was analyzed at the DNA level as a representative species of the subgenus Drosophila. The genome of this species has a single Gapdh gene which is localized at a cytogenetic position likely to be homologous to Gapdh-43 E of D. melanogaster. Comparison of its sequence with the sequence of the D. melanogaster Gapdh genes indicates that the two genes of D. melanogaster are more similar to one another than either is to the gene from D. hydei. The Gapdh gene from D. hydei contains an intron following codon 29. Neither Gapdh gene of D. melanogaster has an intron within the coding region. Southern blots of genomic DNA were used to determine which species have duplicate Gapdh genomic sequences. Gene amplification was used to determine which species have a Gapdh gene that is interrupted by an intron. Species of the subgenus Drosophila have a single Gapdh gene with an intron. Species of the willistoni and saltans groups have a single Gapdh gene that does not contain an intron.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the long terminal repeats of micropia elements microdissected from the Y-chromosomal lampbrush loops "threads" of Drosophila hydei

Four micropia elements from Drosophila melanogaster and D. hydei have been analysed by sequencing. Two elements, from D. hydei, micropia-DhMiF8 and -DhMiF2, were recovered by cloning microdissected Y-chromosomal lampbrush loops "threads". This method allows isolation of repetitive sequences from defined chromosomal positions, but recovery of large and overlapping inserts is difficult. In case of the Y-chromosomal micropia elements it was not possible to define the endpoints of their long terminal repeat sequences precisely. Comparison of these locus-defined micropia elements to complete micropia elements isolated from D. melanogaster allowed identification of micropia-DhMiF8 and micropia-DhMiF2 long terminal repeats (LTRs). LTR sequences from the two Drosophila species are not conserved except for a few short sequences found at comparable positions that are believed to have functional significance. In contrast, the Leu-tRNA primer binding site and plus strand primer binding site are conserved between D. melanogaster and D. hydei.