The evolution of the vertebrate beta-globin gene promoter

Complexity analysis is capable of highlighting those gross evolutionary changes in gene promoter regions (loosely termed "promoter shuffling") that are undetectable by conventional DNA sequence alignment. Complexity analysis was therefore used here to identify the modular components (blocks) of the orthologous beta-globin gene promoter sequences of 22 vertebrate species, from zebrafish to humans. Considerable variation between the beta-globin gene promoters was apparent in terms of block presence/absence, copy number, and relative location. Some sequence blocks appear to be ubiquitous, whereas others are restricted to a specific taxon. Block similarities were also evident between the promoters of the paralogous human beta-like globin genes. It may be inferred that a wide variety of different mutational mechanisms have operated upon the beta-globin gene promoter over evolutionary time. Because these include gross changes such as deletion, duplication, amplification, elongation, contraction, and fusion, as well as the steady accumulation of single base-pair substitutions, it is clear that some redefinition of the term "promoter shuffling" is required. This notwithstanding, and as previously described for the vertebrate growth hormone gene promoter, the modular structure of the beta-globin promoter region and those of its paralogous counterparts have continually been rearranged into new combinations through the alteration, or shuffling, of preexisting blocks. Some of these changes may have had no influence on promoter function, but others could have altered either the level of gene expression or the responsiveness of the promoter to external stimuli. The comparative study of vertebrate beta-globin gene promoter regions described here confirms the generality of the phenomenon of sequence block shuffling and thus supports the view that it could have played an important role in the evolution of differential gene expression.     

High rate of genetic rearrangement during replication of a Moloney murine leukemia virus-based vector.

A protocol was designed to measure the forward mutation rate over an entire gene replicated as part of a Moloney murine leukemia virus-based vector. For these studies, the herpes simplex virus thymidine kinase (tk) gene under the control of the spleen necrosis virus U3 promoter was used as target sequence since it allows selection for either the functional or the inactivated gene. Our results indicate that after one round of retroviral replication, the tk gene is inactivated at an average rate of 0.08 per cycle of replication. Southern blotting revealed that the majority of the mutant proviruses resulted from gross rearrangements and that deletions of spleen necrosis virus and tk sequences were the most frequent cause of the gene inactivation. Sequence analysis of the mutant proviruses suggested that homologous as well as nonhomologous recombination was involved in the observed rearrangements. Some mutations consisted of simple deletions, and others consisted of deletions combined with insertions. The frequency at which these mutations occurred during one cycle of retroviral replication provides evidence indicating that Moloney murine leukemia virus-based vectors may undergo genetic rearrangement at high rates. The high rate of rearrangement and its relevance for retrovirus-mediated gene transfer are discussed.     

Novel complex genomic rearrangement of the BRCA1 gene

Initial BRCA1 and BRCA2 analyses conducted in breast and ovarian cancer families were focused on identification of mutations in coding sequences and splicing sites of the genes. Large genomic rearrangements as well as mutations in promoter or untranslated regions have been missed by standard detection strategies. Nevertheless, in Western countries, a detailed study of families with strong linkage to BRCA1 identified large genomic deletions and rearrangements in this gene as early as 1997. To date, no such gene alteration has been described in Central and Eastern European populations. In our study of BRCA1/2 genes in the Czech population, we have detected a complex genomic rearrangement in BRCA1 using RNA-based analysis for mutation screening. This rearrangement involves exons 21 and 22 and results in a protein product lacking BRCT domain important for its function.     

Chromosomal aberrations induced by double strand DNA breaks

It has been suggested that introduction of double strand DNA breaks (DSBs) into mammalian chromosomes can lead to gross chromosomal rearrangements through improper DNA repair. To study this phenomenon, we employed a model system in which a double strand DNA break can be produced in human cells in vivo at a predetermined location. The ensuing chromosomal changes flanking the breakage site can then be cloned and characterized. In this system, the recognition site for the I-SceI endonuclease, whose 18 bp recognition sequence is not normally found in the human genome, is placed between a strong constitutive promoter and the Herpes simplex virus thymidine kinase (HSV-tk) gene, which serves as a negative selectable marker. We found that the most common mutation following aberrant DSB repair was an interstitial deletion; these deletions typically showed features of non-homologous end joining (NHEJ), such as microhomologies and insertions of direct or inverted repeat sequences. We also detected more complex rearrangements, including large insertions from adjacent or distant genomic regions. The insertion events that involved distant genomic regions typically represented transcribed sequences, and included both L1 LINE elements and sequences known to be involved in genomic rearrangements. This type of aberrant repair could potentially lead to gene inactivation via deletion of coding or regulatory sequences, or production of oncogenic fusion genes via insertion of coding sequences.     

Rearrangement rate following the whole-genome duplication in teleosts

It is now clear that a whole-genome duplication (WGD) occurred at the base of the teleost fish lineage. Like the other anciently polyploid genomes investigated so far, teleost genomes now behave like diploids with chromosomes forming pairs at meiosis. The diploidization process is currently poorly understood. It is associated with many gene deletions, such that one of the duplicates is lost at most loci and has also been proposed to coincide with an increase in genomic instability. Here we ask whether WGD is a determinant of the genomic rearrangement rate in teleosts. We study variability of the rates of rearrangement along a vertebrate phylogenetic tree, composed of 3 tetrapods (human, chicken, and mouse) and 3 teleost fishes (zebrafish, Tetraodon, and Takifugu), whose complete genome sequences are available. We devise a simple parsimony method for counting rearrangements, which takes into account various methodological complications caused by the WGD and the subsequent gene losses. We show that there does appear to be an increase in rearrangement rate after WGD, but that there is also a great deal of additional variability in rearrangement rates across species.     

脊椎动物线粒体DNA的基因重排

将GenBank上已公布的321种脊椎动物mtDNA全序列,按纲整理归类,绘制基因排布图并进行比对。比对结果表明：81个物种的mtDNA中观察到基因重排现象,涉及脊椎动物各纲,其中9个物种同时存在基因顺序变化和基因倒置现象,所有的基因重排都涉及tRNA的变化。脊椎动物mtDNA基因顺序变化可分为3类：1)邻接的基因或片段的位置交换;2)接近于控制序列或轻链起始位点的基因或片段的位置变化,有时还伴随着控制序列的倍增;3)I-Q-M区域的变化。所有鸟类、蛇类、鳄类和有袋类的mtDNA具有各自独特的基因排列顺序。基因倒置现象常见于鱼类和哺乳类,且多表现为tRNA从轻链往重链上迁移。本文就这些基因重排现象、发生重排的机制和mtDNA基因重排在系统发生研究中的应用做一简要概述。     

Evolution of the proximal promoter region of the mammalian growth hormone gene.

The evolutionary relationship between the proximal growth hormone (GH) gene promoter sequences of 12 mammalian species was explored by comparison of their trinucleotide composition and by multiple sequence alignment. Both approaches yielded results that were consistent with the known fossil record-based phylogeny of the analysed sequences, suggesting that the two methods of tree reconstruction might be equally efficient and reliable. The pattern of evolution inferred for the mammalian GH gene promoters was found to vary both temporally and spatially. Thus, two distinct regions devoid of any evolutionary changes exist in primates, but only one of these 'gaps' is also observed in rodents, and neither is seen in ruminants. Furthermore, different evolutionary rates must have prevailed during different periods of evolutionary time and in different lineages, with a dramatic increase in evolutionary rate apparent in primates. Since a similar pattern of discontinuity has been previously noted for the evolution of the GH-coding regions, it may reflect the action of positive selection operating upon the GH gene as a single cohesive unit. Strong evidence for the action of gene conversion between primate GH gene promoters is provided by the fact that the human GH1 and GH2 sequences, which are thought to have diverged before the divergence of Old World monkeys from great apes, are more similar to one another than either is to the rhesus monkey GH2 promoter. Finally, it was noted that a number of nucleotide positions in the GH1 gene promoter that are polymorphic in humans appear to be highly conserved in mammals. This apparent conundrum, which could represent a caveat for the interpretation of phylogenetic footprinting studies, is potentially explicable in terms either of reduced genetic diversity in highly inbred animal species or insufficient population data from non-human species.     

Multiple sequence rearrangements accompanying the duplication of a tRNAPro gene in wheat mitochondrial DNA

In the course of isolating tRNA genes from wheat mtDNA, we have found the same tRNA^Pro gene in two different Hind III restriction fragments, H-P1 (0.7 kbp) and H-P2 (1.7 kbp). Sequences immediately flanking these duplicate genes are closely related, although not identical; sequence comparisons suggest that multiple rearrangements have occurred in the vicinity of the H-P2 tRNA^Pro gene, relative to the H-P1 version. The chimeric nature of H-P2 is emphasized by the presence of sequences that are also found upstream of the wheat mitochondrial 26S rRNA gene, as well as sequences derived from chloroplast DNA. Comparison of H-P2 with H-P1 plus upstream sequences provides some insight into possible molecular events that might have generated H-P2. In particular, such comparisons suggest a model in which the homologous sequences in H-P2 are seen to be derived from H-P1 plus upstream sequences as a result of an intragenomic, site-specific rearrangement event, followed by amplification of the product, its fixation in the mitochondrial genome, and subsequent sequence divergence (single base changes as well as insertions/deletions of up to 50 nucleotides). The results reported here implicate particular primary sequence motifs in certain of the rearrangements that characterize H-P2.     

Chromosome evolution in eukaryotes: a multi-kingdom perspective

In eukaryotes, chromosomal rearrangements, such as inversions, translocations and duplications, are common and range from part of a gene to hundreds of genes. Lineage-specific patterns are also seen: translocations are rare in dipteran flies, and angiosperm genomes seem prone to polyploidization. In most eukaryotes, there is a strong association between rearrangement breakpoints and repeat sequences. Current data suggest that some repeats promoted rearrangements via non-allelic homologous recombination, for others the association might not be causal but reflects the instability of particular genomic regions. Rearrangement polymorphisms in eukaryotes are correlated with phenotypic differences, so are thought to confer varying fitness in different habitats. Some seem to be under positive selection because they either trap favorable allele combinations together or alter the expression of nearby genes. There is little evidence that chromosomal rearrangements cause speciation, but they probably intensify reproductive isolation between species that have formed by another route.     

Genomic rearrangements of the APC tumor-suppressor gene in familial adenomatous polyposis

Germline mutations of the adenomatous polyposis coli (APC) tumor-suppressor gene result in the hereditary colorectal cancer syndrome familial adenomatous polyposis (FAP). Almost all APC mutations that have been identified are single-nucleotide alterations, small insertions, or small deletions that would truncate the protein product of the gene. No well-characterized intragenic rearrangement of APC has been described, and the prevalence of this type of mutation in FAP patients is not clear. We screened 49 potential FAP families and identified 26 different germline APC mutations in 30 families. Four of these mutations were genomic rearrangements resulting from homologous and nonhomologous recombinations mediated by Alu elements. Two of these four rearrangements were complex, involving deletion and insertion of nucleotides. Of these four rearrangements, one resulted in the deletion of exons 11 and 12 and two others resulted in either complete or partial deletion of exon 14. The fourth rearrangement grossly altered the sequence within intron 14. Although this rearrangement did not affect any coding sequence of APC at the genomic DNA level, it caused inappropriate splicing of exon 14. These rearrangements were initially revealed by analyzing cDNAs and could not have been identified by using mutation detection methods that screened each exon individually. The identification of a rearrangement that did not alter any coding exons yet affected the splicing further underscores the importance of using cDNA for mutation analysis. The identification of four genomic rearrangements among 30 mutations suggests that genomic rearrangements are frequent germline APC mutations.     

Aberrant transposition of a Tc1-mariner element, impala, in the fungus Fusarium oxysporum

We previously determined that the impalaD transposable element of Fusarium oxysporum was able to mobilize a non autonomous copy of impala ( niaD::imp::hph), inserted in the niaD gene encoding nitrate reductase. Generally, mobilization results in the recovery of Nia(+) revertants at low frequency. In the course of this study, we recovered a transformant that gave rise to Nia(+) revertants at a high rate. These revertants displayed atypical phenotypes and showed a niaD hybridization pattern different from that in more typical revertants. Molecular analysis of the structure of the transformant and atypical revertants indicated that (i) in the transformant, two copies of impala, one defective and one active, were inserted at the same genomic locus in a head-to-head orientation; and (ii) all the revertants analyzed presented the same chromosomal rearrangement, an inversion resulting in the replacement of the niaD promoter by a new sequence containing a cryptic promoter. We also frequently observed additional DNA rearrangements (deletion or inversion) in these revertants. The sequences at the rearrangement junctions indicated the occurrence of a transposition event that used the ITRs (Inverted Terminal Repeats) of separate transposons arranged in direct orientation. These features can be interpreted as the consequences of an aberrant transposition process. Such a process may account for the rearrangements observed in some genomic regions containing multiple transposon ends, and could serve as a mechanism for the generation of genetic diversity.     

Multiple origins and rapid evolution of duplicated mitochondrial genes in parthenogenetic geckos (Heteronotia binoei; Squamata, Gekkonidae)

Accumulating evidence for alternative gene orders demonstrates that vertebrate mitochondrial genomes are more evolutionarily dynamic than previously thought. Several lineages of parthenogenetic lizards contain large, tandem duplications that include rRNA, tRNA, and protein-coding genes, as well as the control region. Such duplications are hypothesized as intermediate stages in gene rearrangement, but the early stages of their evolution have not been previously studied. To better understand the evolutionary dynamics of duplicated segments of mitochondrial DNA, we sequenced 10 mitochondrial genomes from recently formed ( approximately 300,000 years ago) hybrid parthenogenetic geckos of the Heteronotia binoei complex and 1 from a sexual form. These genomes included some with an arrangement typical of vertebrates and others with tandem duplications varying in size from 5.7 to 9.4 kb, each with different gene contents and duplication endpoints. These results, together with phylogenetic analyses, indicate independent and frequent origins of the duplications. Small, direct repeats at the duplication endpoints imply slipped-strand error as a mechanism generating the duplications as opposed to a false initiation/termination of DNA replication mechanism that has been invoked to explain duplications in other lizard mitochondrial systems. Despite their recent origin, there is evidence for nonfunctionalization of genes due primarily to deletions, and the observed pattern of gene disruption supports the duplication-deletion model for rearrangement of mtDNA gene order. Conversely, the accumulation of mutations between these recent duplicates provides no evidence for gene conversion, as has been reported in some other systems. These results demonstrate that, despite their long-term stasis in gene content and arrangement in some lineages, vertebrate mitochondrial genomes can be evolutionary dynamic even at short timescales.     

DNA sequences 3' of the Ig H chain cluster rearrange in mouse B cell lines

A mouse myeloma cell line MPC11 (IgG2b, kappa) and variants derived from it have been used to study DNA rearrangements that occur at the Ig H chain locus. One variant, F5.5, has acquired both VH gene and C epsilon gene rearrangements. Through genomic Southern blot analysis initially directed to mapping the C epsilon gene rearrangement, we observed that the VH region rearrangement was linked, through an inversion event, to sequences that originate 3' of the CH cluster, i.e., 3' of the C alpha gene. Subsequent studies have shown that DNA rearrangements within the region 3' of the C alpha gene are detected in several other mouse myeloma and hybridoma cell lines and are not associated with the expression of specific isotypes.