In vitro methylation of CpG-rich islands.

CpG islands are distinguishable from the bulk of vertebrate DNA for being unmethylated and CpG-rich. Since CpG doublets are the specific target of eukaryotic DNA methyltransferases, CpG-rich sequences might be expected to be good methyl-accepting substrates in vitro, despite their unmethylated in vivo condition. This was tested using a partially purified DNA-methyltransferase from human placenta and several cloned CpG-rich or CpG-depleted sequences. The efficiency of methylation was found to be proportional to the CpG content for CpG-depleted regions, which are representative of the bulk genome. However, methylation was much less efficient for CpG frequencies higher than 1 in 12 nucleotides, reaching only 60% of the expected level. That suggests that the close CpG spacing typical of CpG-islands somehow inhibits mammalian DNA methyltransferase. The implications of these findings on the in vivo pattern of DNA methylation are discussed.     

Fixation of the human-specific CMP-N-acetylneuraminic acid hydroxylase pseudogene and implications of haplotype diversity for human evolution

The human CMP-N-acetylneuraminic acid hydroxylase gene (CMAH) suffered deletion of an exon that encodes an active center for the enzyme approximately 3.2 million years ago (MYA). We analyzed a 7.3-kb intronic region of 132 CMAH genes to explore the fixation process of this pseudogene and the demographic implication of its haplotype diversity. Fifty-six variable sites were sorted into 18 different haplotypes with significant linkage disequilibrium. Despite the rather low nucleotide diversity, the most recent common ancestor at CMAH dates to 2.9 MYA. This deep genealogy follows shortly after the original exon deletion, indicating that the deletion has fixed in the population, although whether this fixation was facilitated by natural selection remains to be resolved. Remarkable features are exceptionally long persistence of two lineages and the confinement of one lineage in Africa, implying that some African local populations were in relative isolation while others were directly involved in multiple African exoduses of the genus Homo. Importantly, haplotypes found in Eurasia suggest interbreeding between then-contemporaneous human species. Although population structure within Africa complicates the interpretation of phylogeographic information of haplotypes, the data support a single origin of modern humans, but not with complete replacement of archaic inhabitants by modern humans.     

A maximum likelihood method for analyzing pseudogene evolution: implications for silent site evolution in humans and rodents.

We present a new likelihood method for detecting constrained evolution at synonymous sites and other forms of nonneutral evolution in putative pseudogenes. The model is applicable whenever the DNA sequence is available from a protein-coding functional gene, a pseudogene derived from the protein-coding gene, and an orthologous functional copy of the gene. Two nested likelihood ratio tests are developed to test the hypotheses that (1) the putative pseudogene has equal rates of silent and replacement substitutions; and (2) the rate of synonymous substitution in the functional gene equals the rate of substitution in the pseudogene. The method is applied to a data set containing 74 human processed-pseudogene loci, 25 mouse processed-pseudogene loci, and 22 rat processed-pseudogene loci. Using the informatics resources of the Human Genome Project, we localized 67 of the human-pseudogene pairs in the genome and estimated the GC content of a large surrounding genomic region for each. We find that, for pseudogenes deposited in GC regions similar to those of their paralogs, the assumption of equal rates of silent and replacement site evolution in the pseudogene is upheld; in these cases, the rate of silent site evolution in the functional genes is approximately 70% the rate of evolution in the pseudogene. On the other hand, for pseudogenes located in genomic regions of much lower GC than their functional gene, we see a sharp increase in the rate of silent site substitutions, leading to a large rate of rejection for the pseudogene equality likelihood ratio test.     

A gene fusion at a homeobox locus: alterations in leaf shape and implications for morphological evolution.

Compound leaves are seen in many angiosperm genera and are thought to be either fundamentally different from simple leaves or elaborations of simple leaves. The knotted1-like homeobox (knox) genes are known to regulate plant development. When overexpressed in homologous or heterologous species, this family of genes can cause changes in leaf morphology, including excessive leaf compounding in tomato. We describe here an instance of a spontaneously arisen fusion between a gene encoding a metabolic enzyme and a homeodomain protein. We show that the fusion results in overexpression of the homeodomain protein and a change in morphology that approximates the changes caused by overexpression of the same gene under the control of the cauliflower mosaic virus 35S promoter in transgenic plants. Exon-shuffling events can account for the modularity of proteins. If the shuffled exons are associated with altered promoters, changes in gene expression patterns can result. Our results show that gene fusions of this nature can cause changes in expression patterns that lead to altered morphology. We suggest that such phenomena may have played a role in the evolution of form.     

The human serum amyloid A locus SAA4 is a pseudogene.

We have isolated the human genomic DNA clone GSAA4 from a size-selected Bgl II library by hybridization to a probe derived from the human serum amyloid A gene GSAA1. Sequencing the 5' end of this clone revealed a region similar to the first exon of gene GSAA1 but with significant nucleotide differences and mutation of the 3' splice site. The restriction map of the GSAA4 clone corresponds to that for the locus "SAA4" recently reported by others. Sequence and hybridization details indicate that the locus in clone GSAA4 is a member of the human serum amyloid A gene family and contains a pseudogene. Isolating GSAA4 completes the collection of clones needed to account for all bands found in blot hybridizations of human DNA using serum amyloid A gene probes.     

The human glucocerebrosidase gene and pseudogene: structure and evolution

We report the sequence of the entire human gene encoding beta-glucocerebrosidase and that of the associated pseudogene. The gene contains 11 exons extending from base pair 355 to base pair 7232 in the overall sequence. The gene promoter contains TATA- and CAT-like boxes upstream of the major 5' end of the glucocerebrosidase RNA. The two TATA boxes lie between nucleotides (-23)-(-27) and (-33)-(-39) and the two possible CAT boxes reside between nucleotides (-90)-(-94) and (-96)-(-99) in relation to the major 5' end of the mRNA. The functionality of the promoter region was monitored by coupling it to the bacterial gene coding for chloramphenicol acetyltransferase (CAT) and assaying the expression of the enzyme in cells transfected with this vector. The glucocerebrosidase promoter not only directs synthesis of the bacterial enzyme but also exhibits the same pattern of tissue-specific expression as that of the endogenous gene. An apparently tightly linked pseudogene is approximately 96% homologous to the functional gene. However, introns 2, 4, 6, and 7 have large "deletions" consisting of Alu sequences 313, 626, 320, and 277 bp in length, respectively. It is entirely possible that the ancestral gene lacks these sequences and that they have been inserted into the introns of the functioning gene. There is also a 55-bp deletion from a part of exon 9 flanked by a short inverted repeat. The sequence data should facilitate development of methods for diagnosis of Gaucher disease at the molecular level.     

A pseudogene for human glutathione peroxidase.

Glutathione peroxidases (GPx) serve a bioprotective function in the reduction of peroxides to less toxic substances. Both cellular and secreted forms of the protein have been reported, as well a number of distinct cDNA sequences. Previous efforts have described three distinct loci on human chromosomes 3, 21 and X which hybridize to a GPX cDNA and these authors have speculated that only the chromosome 3 locus encodes a functional GPX gene. This conclusion was based on mapping studies showing a precise deletion of intron sequences in the GPX loci on chromosomes 21 and X despite strong conservation among these sequences in both the coding and 3'-untranslated regions. To pursue this issue, we have isolated the chromosome 21 GPX locus by molecular cloning and determined its nucleotide sequence. Consistent with the expectations of McBride et al. [Biofactors 4 (1988) 285-292], the sequence does reveal a highly conserved processed pseudogene. It is suggested that a retrotransposed copy of the GPX gene integrated into chromosome 21 and may have maintained activity prior to the accumulation of inactivating mutations.     

Short tandem-repeat polymorphism/alu haplotype variation at the PLAT locus: implications for modern human origins

Two dinucleotide short tandem-repeat polymorphisms (STRPs) and a polymorphic Alu element spanning a 22-kb region of the PLAT locus on chromosome 8p12-q11.2 were typed in 1,287-1,420 individuals originating from 30 geographically diverse human populations, as well as in 29 great apes. These data were analyzed as haplotypes consisting of each of the dinucleotide repeats and the flanking Alu insertion/deletion polymorphism. The global pattern of STRP/Alu haplotype variation and linkage disequilibrium (LD) is informative for the reconstruction of human evolutionary history. Sub-Saharan African populations have high levels of haplotype diversity within and between populations, relative to non-Africans, and have highly divergent patterns of LD. Non-African populations have both a subset of the haplotype diversity present in Africa and a distinct pattern of LD. The pattern of haplotype variation and LD observed at the PLAT locus suggests a recent common ancestry of non-African populations, from a small population originating in eastern Africa. These data indicate that, throughout much of modern human history, sub-Saharan Africa has maintained both a large effective population size and a high level of population substructure. Additionally, Papua New Guinean and Micronesian populations have rare haplotypes observed otherwise only in African populations, suggesting ancient gene flow from Africa into Papua New Guinea, as well as gene flow between Melanesian and Micronesian populations.     

Cultural evolution: implications for understanding the human language faculty and its evolution

Human language is unique among the communication systems of the natural world: it is socially learned and, as a consequence of its recursively compositional structure, offers open-ended communicative potential. The structure of this communication system can be explained as a consequence of the evolution of the human biological capacity for language or the cultural evolution of language itself. We argue, supported by a formal model, that an explanatory account that involves some role for cultural evolution has profound implications for our understanding of the biological evolution of the language faculty: under a number of reasonable scenarios, cultural evolution can shield the language faculty from selection, such that strongly constraining language-specific learning biases are unlikely to evolve. We therefore argue that language is best seen as a consequence of cultural evolution in populations with a weak and/or domain-general language faculty.     

Erbb is linked to the alpha-globin locus on mouse chromosome 11.

A fragment of the human gene for c-erb-B was used to map homologous sequences in mice. Analysis of somatic cell hybrids and recombinant inbred and congenic mouse strains indicated that this gene, designated Erbb, is closely linked to the gene for alpha-globin on mouse chromosome 11. Several genes controlling hematopoietic differentiation map to mouse chromosome 11.     

Association of two DNA polymorphisms in the alpha-globin gene cluster: implications for genetic analysis.

A new polymorphic BgI II restriction endonuclease site in the alpha-globin gene complex has been found in Cypriot, Sardinian, and Greek populations. In all cases, this polymorphism is linked to a particular hypervariable region between the zeta 2 and zeta 1 genes. This suggests that these hypervariable regions are stable and will be useful for genetic analysis.     

An axonemal dynein at the Hybrid Sterility 6 locus: implications for t haplotype-specific male sterility and the evolution of species barriers

Poor sperm motility characterized by a distinct aberration in flagellar waveform known as ``curlicue' is a hallmark of t haplotype (t) homozygous male sterility. Previous studies have localized ``curlicue' and a flagellar developmental defect, ``whipless', to the Hybrid Sterility 6 locus (Hst6), between the markers Pim1 and Crya1. More recent heterospecific breeding experiments between Mus spretus (Spretus) and Mus musculus domesticus (Domesticus) have mapped the primary source(s) of both ``curlicue' and ``whipless' to a small sub-locus of Hst6, Curlicue a (Ccua). Here we report the complete physical isolation of the Ccua locus and the identification of a candidate gene for expression of both ``whipless' and ``curlicue' at its proximal end, an axonemal dynein heavy chain gene, Dnahc8, formerly mapped by interspecific backcross analysis near Pim1. Dnahc8 mRNA expression commences in the Domesticus wild-type testis just prior to flagellar assembly and is testis-specific in the adult male. However, expression of Dnahc8 is not readily evident in the testis of either Spretus or ``whipless' animals (Domesticus males homozygous for the Spretus allele of Dnahc8). Our results argue that Dnahc8 is fundamental to flagellar organization and function in Domesticus, but not Spretus, and suggest that Dnahc8 is integral to both Hst6- and t-specific male infertility. Received: 13 July 1999 / Accepted: 25 August 1999     

Structural analysis of the locus containing the human C-reactive protein gene and its related pseudogene

The gene for human C-reactive protein (CRP) is mapped within a 34-kilobase pair genomic DNA segment identified by chromosome walking through overlapping DNA fragments cloned into a lambda phage library. Within 16 kilobase pairs upstream and downstream of the locus for the authentic CRP gene, only one other sequence homologous to that for CRP could be found. Sequencing analysis indicates this sequence to be a pseudogene with 50-80% region-specific homology. Comparison of the authentic CRP gene cloned from genomic DNA libraries independently prepared from three patients indicates no difference in the 5' and 3' flanking region, promoter region, or coding sequence. Only a polymorphism in the length of the poly(GT) stretch located in the intron is observed. There appears to be only one gene locus and copy per haploid chromosome for the authentic CRP gene and its pseudogene.     

Characterization of the gorilla carboxyl ester lipase locus, and the appearance of the carboxyl ester lipase pseudogene during primate evolution.

In this study we report on the isolation and characterization of the gorilla carboxyl ester lipase gene, CEL, and the corresponding CEL pseudogene. We also report on the age of the CEL pseudogene.The gorilla CEL gene is 10.5kb long and comprises 11exons intervened by introns similar to the situation in man, mouse and rat. The encoded protein is 998amino acids long and includes a 23amino acid-long leader peptide. Comparison of the coding sequence, excluding exon 11, of CEL from gorilla and man reveals a 97% similarity. Exon 11, which encodes the characteristic proline rich repeats, contains 39 repeated units in gorilla compared to 16 in man. A truncated CEL pseudogene, with the same organization as that found in man, is also shown to be present in the gorilla genome. The gorilla CEL pseudogene is 4.9kb in length and consists of 5exons interrupted by introns. Southern analysis of the gorilla CEL locus shows that the locus is arranged in a similar way as in man with the functional CEL gene being the most 5' one.To bring further insight to the events involved in the rearrangement of the CEL locus, genomic Southern analyses were performed across several primates; Homo sapiens, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus and Macaca arctoides. Results presented show that the CEL gene duplication occurred prior to the separation of Hominidae (man, chimpanzee, gorilla and orangutan) from Old World monkeys (macaque). The deletion of the original CEL gene giving rise to the truncated version of the CEL gene seems, however, to be restricted to man and the great apes only.