Structural and evolutionary relationships among five members of the human gamma-crystallin gene family.

We have characterized five human gamma-crystallin genes isolated from a genomic phage library. DNA sequencing of four of the genes revealed that two of them predict polypeptides of 174 residues showing 71% homology in their amino acid sequence; the other two correspond to closely related pseudogenes which contain the same in-frame termination codon at identical positions in the coding sequence. Two of the genes and one of the pseudogenes are oriented in a head-to-tail fashion clustered within 22.5 kilobases. All three contain a TATA box 60 to 80 base pairs upstream of the initiation codon and a highly conserved segment of 44 base pairs in length immediately preceding the TATA box. The two genes and the two pseudogenes are similar in structure: each contains a small 5' exon encoding three amino acids followed by two larger exons that correspond exactly to the two similar structural domains of the polypeptide. The first intron varies from 100 to 110 base pairs, and the second intron ranges from 1 to several kilobases, rendering an overall gene size of 1.7 to 4.5 kilobases. At least one of the two pseudogenes appears to have been functional before inactivation, suggesting that their identical mutation was generated by gene conversion.     

High rate of recent intron gain and loss in simultaneously duplicated Arabidopsis genes

We examined the gene structure of a set of 2563 Arabidopsis thaliana paralogous pairs that were duplicated simultaneously 20-60 MYA by tetraploidy. Out of a total of 23,164 introns in these genes, we found that 10,004 pairs have been conserved and 578 introns have been inserted or deleted in the time since the duplication event. This intron insertion/deletion rate of 2.7 x 10(-3) to 9.1 x 10(-4) per site per million years is high in comparison to previous studies. At least 56 introns were gained and 39 lost based on parsimony analysis of the phylogenetic distribution of these introns. We found weak evidence that genes undergoing intron gain and loss are biased with respect to gene ontology terms. Gene pairs that experienced at least 2 intron insertions or deletions show evidence of enrichment for membrane location and transport and transporter activity function. We do not find any relationship of intron flux to expression level or G + C content of the gene. Detection of a bias in the location of intron gains and losses within a gene depends on the method of measurement: an intragene method indicates that events (specifically intron losses) are biased toward the 3' end of the gene. Despite the relatively recent acquisition of these introns, we found only one case where we could identify the mechanism of intron origin--the TOUCH3 gene has experienced 2 tandem, partial, internal gene duplications that duplicated a preexisting intron and also created a novel, alternatively spliced intron that makes use of a duplicated pair of cryptic splice sites.     

Gene Duplication and Gene Conversion in the Caenorhabditis elegans Genome

A comprehensive analysis of duplication and gene conversion for 7394 Caenorhabditis elegans genes (about half the expected total for the genome) is presented. Of the genes examined, 40% are involved in duplicated gene pairs. Intrachromosomal or cis gene duplications occur approximately two times more often than expected. In general the closer the members of duplicated gene pairs are, the more likely it is that gene orientation is conserved. Gene conversion events are detectable between only 2% of the duplicated pairs. Even given the excesses of cis duplications, there is an excess of gene conversion events between cis duplicated pairs on every chromosome except the X chromosome. The relative rates of cis and trans gene conversion and the negative correlation between conversion frequency and DNA sequence divergence for unconverted regions of converted pairs are consistent with previous experimental studies in yeast. Three recent, regional duplications, each spanning three genes are described. All three have already undergone substantial deletions spanning hundreds of base pairs. The relative rates of duplication and deletion may contribute to the compactness of the C. elegans genome. Received: 30 July 1998 / Accepted: 12 October 1998     

The human serglycin gene. Nucleotide sequence and methylation pattern in human promyelocytic leukemia HL-60 cells and T-lymphoblast Molt-4 cells.

The complete nucleotide sequence of the 16.7-kb human gene that encodes the peptide core (serglycin) of a secretory granule proteoglycan was determined, thus representing the first proteoglycan peptide core gene to be sequenced in its entirety. The exons, intron 1, and intron 2 comprised 7, 53, and 40% of the gene, respectively. Nineteen Alu-repetitive DNA sequences were interspersed in the gene, accounting for 28% of the total nucleotides in intron 1 and 40% of the nucleotides in intron 2. The nucleotide sequence was then used in an examination of the methylation pattern of the human serglycin gene in human promyelocytic leukemia HL-60 cells that contain serglycin mRNA and in T-lymphoblast Molt-4 cells that do not. With polymerase chain reaction methodology, 13 DNA probes of 250-880 base pairs in length were generated that corresponded to unique, non-Alu sequences spaced throughout the entire human serglycin gene. When blots containing genomic DNA digested with HpaII or MspI were examined with these genomic probes, it was discovered that the 5'-flanking region and intron 1 of the serglycin gene in HL-60 cells were both substantially less methylated than intron 2. In contrast, the entire serglycin gene in Molt-4 cells was highly methylated. Because hypomethylated genes generally are transcribed more efficiently than hypermethylated genes, the high level of serglycin mRNA in HL-60 cells probably is a consequence of the low level of methylation of intron 1 and the 5'-flanking region of the serglycin gene in these cells.     

Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system.

Plasmids containing heteroallelic copies of the Saccharomyces cerevisiae HIS3 gene undergo intramolecular gene conversion in mitotically dividing S. cerevisiae cells. We have used this plasmid system to determine the minimum amount of homology required for gene conversion, to examine how conversion tract lengths are affected by limited homology, and to analyze the role of flanking DNA sequences on the pattern of exchange. Plasmids with homologous sequences greater than 2 kilobases have mitotic exchange rates as high as 2 x 10(-3) events per cell per generation. As the homology is reduced, the exchange rate decreases dramatically. A plasmid with 26 base pairs (bp) of homology undergoes gene conversion at a rate of approximately 1 x 10(-10) events per cell per generation. These studies have also shown that an 8-bp insertion mutation 13 bp from a border between homologous and nonhomologous sequences undergoes conversion, but that a similar 8-bp insertion 5 bp from a border does not. Examination of independent conversion events which occurred in plasmids with heteroallelic copies of the HIS3 gene shows that markers within 280 bp of a border between homologous and nonhomologous sequences undergo conversion less frequently than the same markers within a more extensive homologous sequence. Thus, proximity to a border between homologous and nonhomologous sequences shortens the conversion tract length.     

Genetic Hitchhiking and the Evolution of Reduced Genetic Activity of the Y Sex Chromosome

We report experiments designed to test homology dependence for gene conversion between duplicated chromosomal sequences in cultured mammalian cells. The experimental system is such that gene conversion events not associated with reciprocal exchange are recoverable. For this study four plasmids were constructed. Each contains a different duplication of the herpes simplex virus thymidine kinase (HSV tk) gene sequence. In particular, the interacting sequences share different lengths of homology. Our results indicate that for shared homologies between 295 base pairs (bp) and 1.8 kilobase pairs (kbp) in length, conversion is efficient with the rate being directly proportional to the extent of homology. In contrast, conversion with either 200 bp or 95 bp of homology is inefficient, and the rate is reduced at least seven- or 100-fold, respectively, relative to that observed with 295 bp of homology. These results are consistent with the notion that greater than 200 bp of homology are required for efficient gene conversion between repeated chromosomal sequences in mammalian cells.     

Functional analysis of elements affecting expression of the beta-actin gene of carp.

Regulatory regions of the beta-actin gene of the common carp (Cyprinus carpio) have been examined by linking upstream, 5'-flanking sequences and regions of the first intron to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. By analysis of the mRNA products and encoded CAT activity, we have identified four putative regions that influence expression: (i) a negative regulatory region 2,300 to 1,100 base pairs (bp) ahead of the gene; (ii) a proximal promoter element, containing the highly conserved CCAAT, CC(A/T)6GG, and TATA boxes, that is within the first 204 bp upstream of the initiation site; (iii) a negative element of 426 bp in the 5' region of the first intron; and (iv) a positive 304-bp element near the end of the first intron that contains highly conserved sequences found in all characterized beta-actin genes. The positive intron element is not a classical enhancer; it is position and orientation dependent, as has been observed in other housekeeping genes in vertebrates. Depending on the elements joined together, CAT gene expression can be modulated more than 500-fold in transfected mouse cells.     

Comparison of Bombyx mori and Helicoverpa armigera cytoplasmic actin genes provides clues to the evolution of actin genes in insects

The cytoplasmic actin genes BmA3 and BmA4 of Bombyx mori were found clustered in a single genomic clone in the same orientation. As a similar clustering of the two cytoplasmic actin genes Ha3a and Ha3b also occurs in another lepidopteran, Helicoverpa armigera, we analyzed the sequence of the pair of genes from each species. Due to the high conservation of cytoplasmic actins, the coding sequence of the four genes was easily aligned, allowing the detection of similarities in noncoding exon and intron sequences as well as in flanking sequences. All four genes exhibited a conserved intron inserted in codon 117, an original position not encountered in other species. It can thus be postulated that all of these genes derived from a common ancestral gene carrying this intron after a single event of insertion. The comparison of the four genes revealed that the genes of B. mori and H. armigera are related in two different ways: the coding sequence and the intron that interrupts it are more similar between paralogous genes within each species than between orthologous genes of the two species. In contrast, the other (noncoding) regions exhibited the greatest similarity between a gene of one species and a gene of the other species, defining two pairs of orthologous genes, BmA3 and HaA3a on one hand and BmA4 and HaA3b on the other. However, in each species, the very high similarities of the coding sequence and of the single intron that interrupts it strongly suggest that gene conversion events have homogenized this part of the sequence. As the divergence of the B. mori genes was higher than that of the H. armigera genes, we postulated that the gene conversion occurred earlier in the B. mori lineage. This leads us to hypothesize that gene conversion could also be responsible for the original transfer of the common intron to the second gene copy before the divergence of the B. mori and H. armigera lineages.     

Near intron positions are reliable phylogenetic markers: an application to holometabolous insects

Today, the reconstruction of the organismal evolutionary tree is based mainly on molecular sequence data. However, sequence data are sometimes insufficient to reliably resolve in particular deep branches. Thus, it is highly desirable to find novel, more reliable types of phylogenetic markers that can be derived from the wealth of genomic data. Here, we consider the gain of introns close to older preexisting ones. Because correct splicing is impeded by very small exons, nearby pairs of introns very rarely coexist, that is, the gain of the new intron is nearly always associated with the loss of the old intron. Both events may even be directly connected as in cases of intron migration. Therefore, it should be possible to identify one of the introns as ancient (plesiomorphic) and the other as novel (derived or apomorphic). To test the suitability of such near intron pairs (NIPs) as a marker class for phylogenetic analysis, we undertook an analysis of the evolutionary positions of bees and wasps (Hymenoptera) and beetles (Coleoptera) in relation to moths (Lepidoptera) and dipterans (Diptera) using recently completed genome project data. By scanning 758 putatively orthologous gene structures of Apis mellifera (Hymenoptera) and Tribolium castaneum (Coleoptera), we identified 189 pairs of introns, one from each species, which are located less than 50 nt from each other. A comparison with genes from 5 other holometabolan and 9 metazoan outgroup genomes resulted in 22 shared derived intron positions found in beetle as well as in butterflies and/or dipterans. This strongly supports a basal position of hymenopterans in the holometabolous insect tree. In addition, we found 31 and 12 intron positions apomorphic for A. mellifera and T. castaneum, respectively, which seem to represent changes inside these branches. Another 12 intron pairs indicate parallel intron gains or extraordinarily small exons. In conclusion, we show here that the analysis of phylogenetically nested, nearby intron pairs is suitable to identify evolutionarily younger intron positions and to determine their relative age, which should be of equal importance for the understanding of intron evolution and the reconstruction of the eukaryotic tree.     

Patterns of Sequence Divergence in Daphniid Hemoglobin Genes

In common with other multigene families, sequence diversity in the hemoglobin genes of cladoceran crustaceans has been heavily impacted by gene conversion events. Because of their structural complexity (six exons, five introns), these genes provide a good opportunity to study the influence of intron length and position on the conversion process. This study surveys the patterns of divergence in variants of one hemoglobin gene (H1) from two closely related species of Daphnia using a PCR-based approach. Although its effects were most pronounced at their 5' ends, intron and exon regions of these genes showed similar exposure to gene conversion, excepting intron 2. This intron, which was the only one with a marked length difference among variants, showed substantial sequence divergence, suggesting that gene conversion was disrupted. These results, together with those on hemoglobin gene families in other organisms, indicate that sequence tracts showing gene conversion are often distributed in a mosaic fashion. The reactivation of gene conversion downstream of a block protected from its effects suggests that there are multiple initiation points, and the distribution of conversion tracts suggests that exon/intron splice sites are important in this regard.     

In vitro site-directed mutagenesis with synthetic DNA oligonucleotides yields unexpected deletions and insertions at high frequency.

We have used in vitro site-directed mutagenesis with synthetic DNA oligonucleotides to introduce single nucleotide mutations in yeast mtDNA. In addition to the expected DNA alterations we also recovered with high frequency mutants with large deletions and insertions which arose through interaction with the synthetic DNA fragment. Characterization of a number of these by DNA sequence analysis has permitted reconstruction of the mutagenic events. In all cases, the DNA fragment had base paired with non-adjacent DNA sequences sometimes more than 1000 nucleotides apart from each other on the target strand. The products of such interactions cannot be avoided due to the non-stringent annealing conditions during complementary DNA strand synthesis. However, deliberate mispairing can be directed precisely, as shown by our ability to specifically delete the 1143-bp intron from the yeast mitochondrial gene coding for large ribosomal RNA with a synthetic DNA fragment consisting of the sequence of the exon borders flanking the intron.     

Multicopy single-stranded DNA of Escherichia coli enhances mutation and recombination frequencies by titrating MutS protein

Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA. In Escherichia coli , such molecules are encoded by genetic elements called retrons. The DNA moieties of msDNAs have characteristic stem-loop structures, and most of these structures contain mismatched base pairs. Previously, we showed that retrons encoding msDNAs with mismatched base pairs are mutagenic when present in multicopy plasmids. In this study we show that such msDNAs, in a similar manner to genetic defects in mismatch repair, increase the frequency of interspecies recombination in matings between Salmonella typhimurium and E. coli . To demonstrate interference with mismatch repair by msDNA, we show that the addition of a plasmid containing the gene for MutS protein suppresses the mutagenic and recombinogenic effects of msDNAs. We also show that in mutS mutants, msDNA does not increase the frequency of either mutations or interspecies recombination. We conclude from these findings that the mutagenic and recombinogenic effects of msDNAs are due to titrating out MutS protein.