Population genetics of dinucleotide (dC-dA)n.(dG-dT)n polymorphisms in world populations.

We have characterized eight dinucleotide (dC-dA)n.(dG-dT)n repeat loci located on human chromosome 13q in eight human populations and in a sample of chimpanzees. Even though there is substantial variation in allele frequencies at each locus, at a given locus the most frequent alleles are shared by all human populations. The level of heterozygosity is reduced in isolated or small populations, such as the Pehuenche Indians of Chile, the Dogrib of Canada, and the New Guinea highlanders. On the other hand, larger average heterozygosities are observed in large and cosmopolitan populations, such as the Sokoto population from Nigeria and German Caucasians. Conformity with Hardy-Weinberg equilibrium is generally observed at these loci, unless (a) a population is isolated or small or (b) the repeat motif of the locus is not perfect (e.g., D13S197). Multilocus genotype probabilities at these microsatellite loci do not show departure from the independence rule, unless the loci are closely linked. The allele size distributions at these (CA)n loci do not follow a strict single-step stepwise-mutation model. However, this features does not compromise the ability to detect population affinities, when these loci are used simultaneously. The microsatellite loci examined here are present and, with the exception of the locus D13S197, are polymorphic in the chimpanzees, showing an overlapping distribution of allele sizes with those observed in human populations.     

Genetic mapping of 18s ribosomal RNA-related loci to mouse Chromosomes 5, 6, 9, 12, 17, 18, 19, and X

The organization of ribosomal RNA genes (rDNA) in the genome of the mouse varies significantly from one strain to another, but has been shown to follow the pattern of clusters of tandem repeats located at chromosome ends, often associated with cytological nucleolus organizer regions. The number of copies of the repeat unit at each locus also varies. A probe for the 18S ribosomal RNA sequence on Southern blots reveals both high copy number bands and fainter bands indicative of low repeat number. We have mapped a number of newly identified low-copy-number rDNA loci in C57BL/6J, in addition to placing some of the NOR-associated rDNA repeats on the Jackson interspecific backcross (BSS) map. We suggest that additional low-copy-number loci may remain to be mapped, and that the evolution of rDNA loci in the genome may include the proliferation of single copies by retroinsertion or other mechanisms. Received: 23 February 1996 / Accepted: 29 July 1996     

Primary structure of the goat beta-globin locus control region

The goat beta-globin cluster is composed of a triplicated four-gene set. A locus control region (LCR) containing elements homologous to 5'DNase I hypersensitive sites (HS) 1, 2, and 3 of the human beta-globin LCR has been identified at the 5' end of this locus. We determined 10.2 kb of nucleotide sequence from the goat beta-globin locus control region. Self-comparison of this sequence by dot matrix analysis revealed the presence of six complete and three incomplete artiodactyl repeats. A novel repeated element, termed D repeat, was also identified. Southern blotting analysis demonstrated that these elements exist in the goat genome as a low to medium frequency interspersed repeat family. The absence of any other large region of self-homology (direct or inverted) in the goat LCR suggests that 5'HSs 1, 2, and 3 did not arise through duplication, but rather evolved independently. By comparing goat 5'HS 1 to those of human, rabbit, and mouse, we show a greater than 80% conservation in sequence between the four species. This level of evolutionary conservation suggests that 5'HS 1 plays an important role in the regulation of beta-globin loci.     

Chromosomal localization of human endogenous retroviral element ERV1 to 18q22----q23 by in situ hybridization

From a clone containing the entire locus of human endogenous retroviral element ERV1, we have obtained a DNA probe that is specific for the 3' long terminal repeat (LTR) sequence. This probe was used to map the LTR of ERV1 by in situ hybridization to chromosomes from normal human blood lymphocytes. The LTR was found to be localized to the distal portion of the long arm of human chromosome 18, within bands q22----q23. This chromosome locus is near the constitutive fragile site at band q21.3 on chromosome 18 associated with the 14;18 translocations seen in follicular lymphomas.     

Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein

Endonexin II is a member of the family of Ca2+-dependent phospholipid binding proteins known as annexins. We cloned human endonexin II cDNA and expressed it in Escherichia coli. The apparent size and Ca2+-dependent phospholipid binding properties of purified recombinant endonexin II were indistinguishable from those of the placental protein. A single mRNA of approximately 1.6 kilobase pairs was found to be expressed in human cell lines and placenta and was in close agreement with the length of the cDNA clone (1.59 kilobase pairs). The cDNA predicted a 320-amino acid protein with a sequence that was in agreement with the previously determined partial amino acid sequence of endonexin II isolated from placenta. Endonexin II contained 58, 46, and 43% sequence identity to protein II, calpactin I (p36, protein I), and lipocortin I (p35), respectively. The partial sequence of bovine endonexin I was aligned with the sequence of endonexin II to give 63% sequence identity. Like these other proteins, endonexin II had a 4-fold internal repeat of approximately 70 residues preceded by an amino-terminal domain lacking similarity to the repeated region. It also had significant sequence identity with 67-kDa calelectrin (p68), a protein with an 8-fold internal repeat. Comparing the amino-terminal domains of these four proteins of known sequence revealed that, in general, only endonexin II and protein II had significant sequence identity (29%). Endonexin II was not phosphorylated by Ca2+/phospholipid-dependent enzyme (protein kinase C) even though it contained a threonine at a position analogous to the protein kinase C phosphorylation sites of lipocortin I, calpactin I, and protein II.     

Human trophoblast and the choriocarcinoma cell line BeWo express a truncated HLA Class I molecule

Extravillous trophoblast from normal human placenta has been shown previously to express an unusual form of HLA class I molecule, as does a choriocarcinoma cell line, BeWo. This molecule has a H chain of approximately 40 kDa and appears to be nonpolymorphic. We have isolated and sequenced a HLA class I cDNA clone, which probably corresponds to this molecule, from a library derived from BeWo. The nucleotide sequence shows a high degree of homology with the published sequence of a genomic clone, HLA 6.0, which is the product of a class I locus other than A, B, or C, (provisionally designated "HLA G"). The expressed product of this locus has not previously been localized. We have used the polymerase chain reaction to demonstrate the presence of similar HLA class I sequences in cDNA from normal extravillous trophoblast. Although there is some nucleotide sequence polymorphism the amino acid sequence of this molecule is conserved. It is therefore unlikely to provoke immune responses even though it is found at the fetal-maternal interface.     

Replication fork dynamics and dynamic mutations: the fork-shift model of repeat instability

Gene-specific repeat instability is responsible for >36 human diseases. Active instability varies in a tissue-, developmental stage- and locus-specific manner and occurs in both proliferative and non-proliferative cells. In proliferative cells, DNA replication can contribute to repeat instability either by switching the direction of replication, which changes the repeat sequence that serves as the lagging-strand template (origin switching), or by shifting the location of the origin of replication without altering the replication direction (origin shifting). We propose that changes in the dynamics of replication-fork progression, or architecture, will alter the location of the repeat within the single-stranded lagging-strand template, thereby influencing instability (fork shifting). The fork-shift model, which does not require origin relocation, is influenced by cis-elements and trans-factors associated with driving and maintaining replication forks. The fork-shift model can explain some of the complex behaviours of repeat instability because it is dynamic and responsive to variations in epigenomic and locus activity.     

Localization of the Homolog of a Mouse Craniofacial Mutant to Human Chromosome 18q11 and Evaluation of Linkage to Human CLP and CPO

The transgene-induced mutation 9257 and the spontaneous mutation twirler cause craniofacial and inner ear malformations and are located on mouse chromosome 18 near the ataxia locusax.To map the human homolog of 9257, a probe from the transgene insertion site was used to screen a human genomic library. Analysis of a cross-hybridizing human clone identified a 3-kb conserved sequence block that does not appear to contain protein coding sequence. Analysis of somatic cell hybrid panels assigned the human locus to 18q11. The polymorphic microsatellite markers D18S1001 and D18S1002 were isolated from the human locus and mapped by linkage analysis using the CEPH pedigrees. The 9257 locus maps close to the centromeres of human chromosome 18q and mouse chromosome 18 at the proximal end of a conserved linkage group. To evaluate the role of this locus in human craniofacial disorders, linkage to D18S1002 was tested in 11 families with autosomal dominant nonsyndromic cleft lip and palate and 3 families with autosomal dominant cleft palate only. Obligatory recombinants were observed in 8 of the families, and negative lod scores from the other families indicated that these disorders are not linked to the chromosome 18 loci.     

The fragile X syndromes

Fragile X syndrome is a leading cause of mental retardation worldwide, with an incidence of approximately one case in 2000 live births. It is amongst the most common of human genetic diseases, and was the first to be associated with an unstable trinucleotide (CGG) repeat sequence. It is also characterized by a chromosomal fragile site which was the first of (now) four such sites to be identified at the molecular level. Each shows very similar features suggesting that other representatives of this type of fragile site will likely involve similar sequences. As with the other unstable trinucleotide repeats, the sequence at the fragile X locus is found to be remarkably unstable upon genetic transmission, however many features differ from the other repeats. As repeat expansion at the fragile X locus results in loss of expression of the co-resident FMR1 gene, the basis for clinical features is best understood in this disorder. Two additional fragile sites in the vicinity have been identified, and at least one of these is associated with mental retardation.     

Complete Genomic Sequence of the Human Retinoblastoma Susceptibility Gene

A 180,388-bp contig encompassing the human retinoblastoma gene was sequenced in its entirety. Partial analysis of the sequence revealed (1) a high (A + T)/(G + C) ratio and a high density of Line-1 (L1) repeat sequences, suggesting that the locus maps to G-bands 13q14.12 or 13q14.2; (2) Alu repeats that are asymmetrically oriented over a region extending 87 kb; (3) an overabundance of non-Alu-associated poly(A) tracts 10 bp or larger oriented in the antisense rather than the sense direction (36 vs 6); (4) an Alu sequence nested within an L1 repeat, indicating that the expansion of L1 repeats predates at least some of the Alu expansions; (5) at least three newly discovered microsatellite polymorphisms, one of which was subsequently found to be identical to a polymorphism in a microsatellite-based linkage map of the human genome published by another group; and (6) the basis of previously discovered intragenic RFLPs. This sequence should enhance studies of this locus and of the organization of the human genome.     

Transgenerational effects from exposure to environmental toxic substances

Exposure of mouse germ cells to radiation and chemicals results in mutation, malformation, cancer and other adverse effects (e.g., functional disorders) in the offspring, though these findings have not been proven in human studies. Environmental toxic substances such as urethane (ethyl carbamate) which had been injected subcutaneously to 50 million people as a co-solvent of analgesics and dioxin (an endocrine disruptor) have been found to be associated with adverse effects in the progeny of mice after parental exposures. There are some reports on congenital malformations in the progeny of fathers who had been exposed to dioxin. However, these substances have not shown mutagenicity in in vitro assay systems such as bacterial systems even with S9, cell transformation assays, etc., in spite of their potent teratogenicity and carcinogenicity in in vivo systems. Urethane was negative in the mouse specific locus test for germ cell mutations, but elicited a significant response at the same loci in the offspring of mice treated during pregnancy. Further, urethane is a mutagen in Drosophila germ cell tests, specifically inducing point mutations. Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) does not induce in vivo somatic mutations in mice and rats. It does not induce chromosomal aberrations when the mouse and/or human sperm are treated, but induces mutations at ESTR (expanded simple tandem repeat) loci in mice at low frequencies and also congenital malformations. In this paper, we first present an overview of the results of our studies on transgenerational effects of these toxic substances, compare the results with those obtained after radiation exposure, and then discuss our subsequent studies to reconcile the problems underlying their mutagenicity, teratogenicity and carcinogenicity.