Analysis of the 9p21.3 sequence associated with coronary artery disease reveals a tendency for duplication in a CAD patient.

Tandem segmental duplications (SDs) greater than 10 kb are widespread in complex genomes. They provide material for gene divergence and evolutionary adaptation, while formation of specific de novo SDs is a hallmark of cancer and some human diseases. Most SDs map to distinct genomic regions termed 'duplication blocks'. SDs organization within these blocks is often poorly characterized as they are mosaics of ancestral duplicons juxtaposed with younger duplicons arising from more recent duplication events. Structural and functional analysis of SDs is further hampered as long repetitive DNA structures are underrepresented in existing BAC and YAC libraries. We applied Transformation-Associated Recombination (TAR) cloning, a versatile technique for large DNA manipulation, to selectively isolate the coronary artery disease (CAD) interval sequence within the 9p21.3 chromosome locus from a patient with coronary artery disease and normal individuals. Four tandem head-to-tail duplicons, each ∼50 kb long, were recovered in the patient but not in normal individuals. Sequence analysis revealed that the repeats varied by 10-15 SNPs between each other and by 82 SNPs between the human genome sequence (version hg19). SNPs polymorphism within the junctions between repeats allowed two junction types to be distinguished, Type 1 and Type 2, which were found at a 2:1 ratio. The junction sequences contained an Alu element, a sequence previously shown to play a role in duplication. Knowledge of structural variation in the CAD interval from more patients could help link this locus to cardiovascular diseases susceptibility, and maybe relevant to other cases of regional amplification, including cancer.

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOSYA, replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

Genome-wide mutation avalanches induced in diploid yeast cells by a base analog or an APOBEC deaminase.

Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis.

Human gamma-satellite DNA maintains open chromatin structure and protects a transgene from epigenetic silencing.

The role of repetitive DNA sequences in pericentromeric regions with respect to kinetochore/heterochromatin structure and function is poorly understood. Here, we use a mouse erythroleukemia cell (MEL) system for studying how repetitive DNA assumes or is assembled into different chromatin structures. We show that human gamma-satellite DNA arrays allow a transcriptionally permissive chromatin conformation in an adjacent transgene and efficiently protect it from epigenetic silencing. These arrays contain CTCF and Ikaros binding sites. In MEL cells, this gamma-satellite DNA activity depends on binding of Ikaros proteins involved in differentiation along the hematopoietic pathway. Given our discovery of gamma-satellite DNA in pericentromeric regions of most human chromosomes and a dynamic chromatin state of gamma-satellite arrays in their natural location, we suggest that gamma-satellite DNA represents a unique region of the functional centromere with a possible role in preventing heterochromatin spreading beyond the pericentromeric region.

Evolutionary diversification of SPANX-N sperm protein gene structure and expression.

The sperm protein associated with nucleus in the X chromosome (SPANX) genes cluster at Xq27 in two subfamilies, SPANX-A/D and SPANX-N. SPANX-A/D is specific for hominoids and is fairly well characterized. The SPANX-N gave rise to SPANX-A/D in the hominoid lineage approximately 7 MYA. Given the proposed role of SPANX genes in spermatogenesis, we have extended studies to SPANX-N gene evolution, variation, regulation of expression, and intra-sperm localization. By immunofluorescence analysis, SPANX-N proteins are localized in post-meiotic spermatids exclusively, like SPANX-A/D. But in contrast to SPANX-A/D, SPANX-N are found in all ejaculated spermatozoa rather than only in a subpopulation, are localized in the acrosome rather than in the nuclear envelope, and are expressed at a low level in several nongametogenic adult tissues as well as many cancers. Presence of a binding site for CTCF and its testis-specific paralogue BORIS in the SPANX promoters suggests, by analogy to MAGE-A1 and NY-ESO-1, that their activation in spermatogenesis is mediated by the programmed replacement of CTCF by BORIS. Based on the relative density of CpG, the more extended expression of SPANX-N compared to SPANX-A/D in nongametogenic tissues is likely attributed to differences in promoter methylation. Our findings suggest that the recent duplication of SPANX genes in hominoids was accompanied by different localization of SPANX-N proteins in post-meiotic sperm and additional expression in several nongonadal tissues. This suggests a corresponding functional diversification of SPANX gene families in hominoids. SPANX proteins thus provide unique targets to investigate their roles in the function of spermatozoa, selected malignancies, and for SPANX-N, in other tissues as well.

Mutational analysis of SPANX genes in families with X-linked prostate cancer.

BACKGROUND: Previous genetic linkage studies identified a locus for susceptibility to prostate cancer called HPCX at Xq27. The candidate region contains two clusters of SPANX genes. The first cluster called SPANX-A/D includes SPANX-A1, SPANX-A2, SPANX-B, SPANX-C, and SPANX-D; the second cluster called SPANX-N includes SPANX-N1, SPANX-N2, SPANX-N3, and SPANX-N4. The SPANX genes encode cancer-testis (CT) specific antigens. Previous studies identified SPANX-B and SPANX-D variants produced by gene conversion events, none of which are associated with X-linked prostate cancer. METHODS: In this study we applied transformation-associated recombination cloning (TAR) in yeast to analyze sequence variations in SPANX-A1, SPANX-A2, and SPANX-C genes that are resided within large chromosomal duplications. A SPANX-N1/N4 cluster was analyzed by a routine PCR analysis. RESULTS: None of the sequence variations in the coding regions of these genes is associated with susceptibility to prostate cancer. CONCLUSIONS: Therefore, genetic variation in the SPANX genes is not the actual target variants explaining HPCX. However, it is possible that they play a modifying role in susceptibility to prostate cancer through complex recombinational interaction.

Dynamic structure of the SPANX gene cluster mapped to the prostate cancer susceptibility locus HPCX at Xq27.

Genetic linkage studies indicate that germline variations in a gene or genes on chromosome Xq27-28 are implicated in prostate carcinogenesis. The linkage peak of prostate cancer overlies a region of approximately 750 kb containing five SPANX genes (SPANX-A1, -A2, -B, -C, and -D) encoding sperm proteins associated with the nucleus; their expression was also detected in a variety of cancers. SPANX genes are >95% identical and reside within large segmental duplications (SDs) with a high level of similarity, which confounds mutational analysis of this gene family by routine PCR methods. In this work, we applied transformation-associated recombination cloning (TAR) in yeast to characterize individual SPANX genes from prostate cancer patients showing linkage to Xq27-28 and unaffected controls. Analysis of genomic TAR clones revealed a dynamic nature of the replicated region of linkage. Both frequent gene deletion/duplication and homology-based sequence transfer events were identified within the region and were presumably caused by recombinational interactions between SDs harboring the SPANX genes. These interactions contribute to diversity of the SPANX coding regions in humans. We speculate that the predisposition to prostate cancer in X-linked families is an example of a genomic disease caused by a specific architecture of the SPANX gene cluster.

The Saccharomyces cerevisiae ESU1 gene, which is responsible for enhancement of termination suppression, corresponds to the 3'-terminal half of GAL11.

A DNA fragment enhancing efficiency of [PSI+]-dependent termination suppressor, sup111, was isolated from a genomic library of Saccharomyces cerevisiae and its function was attributed to an ORF of 1272 bp. This ORF, designated ESU1 (enhancer of termination suppression), corresponded to the 3'-terminal portion of GAL11. Contrasting to ESU1, GAL11 lowered the suppression efficiency of [PSI+] sup111. ESU1 possesses a TATA-like sequence of its own and three ATG codons following it within a distance of about 70 bp and all in the same reading frame as GAL11. A 52.7 kDa protein corresponding in size to the predicted Esu1 protein is detected by western blot analysis using anti-Gal11 antiserum. We therefore conclude that ESU1 is the gene that encodes a polypeptide corresponding to the C-terminal 424 amino acids of Gal11. It was further found that ESU1 increases the level of GAL11 mRNA and probably also of its own mRNA. Moreover, ESU1 increased the cellular level of mRNA transcribed from the leu2-1(UAA) mutant gene, while GAL11 did not. Based on these findings, we propose the following scheme for the events taking place in the [PSI+] sup111 cell that is transformed with an ESU1-bearing plasmid: (a) ESU1 stimulates transcription of leu2-1; (b) leu2-1 mRNA is not effectively degraded because of the possession of sup111, which belongs to the upf group; (c) [PSI+] causes increased mis-termination due to depletion of eRF3; (d) functional Leu2 product is made using leu2-1 mRNA; and (d) suppression of leu2-1 is eventually accomplished.

Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation.

Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. An investigation into the structural and the functional relationship in centromeric tandem repeats in HACs requires the ability to manipulate repeat substructure efficiently. We describe here a new method to rapidly amplify human alphoid tandem repeats of a few hundred base pairs into long DNA arrays up to 120 kb. The method includes rolling-circle amplification (RCA) of repeats in vitro and assembly of the RCA products by in vivo recombination in yeast. The synthetic arrays are competent in HAC formation when transformed into human cells. As short multimers can be easily modified before amplification, this new technique can identify repeat monomer regions critical for kinetochore seeding. The method may have more general application in elucidating the role of other tandem repeats in chromosome organization and dynamics.

The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein.

The most common cause of primary autosomal recessive microcephaly (MCPH) appears to be mutations in the ASPM gene which is involved in the regulation of neurogenesis. The predicted gene product contains two putative N-terminal calponin-homology (CH) domains and a block of putative calmodulin-binding IQ domains common in actin binding cytoskeletal and signaling proteins. Previous studies in mouse suggest that ASPM is preferentially expressed in the developing brain. Our analyses reveal that ASPM is widely expressed in fetal and adult tissues and upregulated in malignant cells. Several alternatively spliced variants encoding putative ASPM isoforms with different numbers of IQ motifs were identified. The major ASPM transcript contains 81 IQ domains, most of which are organized into a higher order repeat (HOR) structure. Another prominent spliced form contains an in-frame deletion of exon 18 and encodes 14 IQ domains not organized into a HOR. This variant is conserved in mouse. Other spliced variants lacking both CH domains and a part of the IQ motifs were also detected, suggesting the existence of isoforms with potentially different functions. To elucidate the biochemical function of human ASPM, we developed peptide specific antibodies to the N- and C-termini of ASPM. In a western analysis of proteins from cultured human and mouse cells, the antibodies detected bands with mobilities corresponding to the predicted ASPM isoforms. Immunostaining of cultured human cells with antibodies revealed that ASPM is localized in the spindle poles during mitosis. This finding suggests that MCPH is the consequence of an impairment in mitotic spindle regulation in cortical progenitors due to mutations in ASPM.

Evolution of the tumor suppressor BRCA1 locus in primates: implications for cancer predisposition.

Germ-line mutations in the BRCA1 gene predispose affected individuals to breast and ovarian cancer syndromes. In an attempt to systematically analyze a broader spectrum of genetic changes ranging from frequent exon deletions and duplications to amino acid replacements and protein truncations, we isolated and characterized full size BRCA1 homologues from a representative group of non-human primates. Our analysis represents the first comprehensive sequence comparison of primate BRCA1 loci and corresponding proteins. The comparison revealed an unusually high proportion of indels in non-coding DNA. The major force driving evolutionary changes in non-coding BRCA1 sequences was Alu-mediated rearrangements, including Alu transpositions and Alu-associated deletions, indicating that structural instability of this locus may be intrinsic in anthropoids. Analysis of the non-synonymous/synonymous ratio in coding portions of the gene revealed the presence of both conserved and rapidly evolving regions in the BRCA1 protein. Previously, a rapidly evolving region with evidence of positive evolutionary selection in human and chimpanzee had been identified only in exon 11. Here, we show that most of the internal BRCA1 sequence is variable between primates and evolved under positive selection. In contrast, the terminal regions of BRCA1, which encode the RING finger and BRCT domains, experienced negative selection, which left them almost identical between the compared primates. Distribution of the reported missense mutations, but not frameshift and nonsense mutations, is positively correlated with BRCA1 protein conservation. Finally, on the basis of protein sequence conservation, we identified missense changes that are likely to compromise BRCA1 function.

A general cloning system to selectively isolate any eukaryotic or prokaryotic genomic region in yeast.

BACKGROUND: Transformation-associated recombination (TAR) cloning in yeast is a unique method for selective isolation of large chromosomal fragments or entire genes from complex genomes. The technique involves homologous recombination, during yeast spheroplast transformation, between genomic DNA and a TAR vector that has short (approximately 60 bp) 5' and 3' gene targeting sequences (hooks). RESULT: TAR cloning requires that the cloned DNA fragment carry at least one autonomously replicating sequence (ARS) that can function as the origin of replication in yeast, which prevents wide application of the method. In this paper, we describe a novel TAR cloning system that allows isolation of genomic regions lacking yeast ARS-like sequences. ARS is inserted into the TAR vector along with URA3 as a counter-selectable marker. The hooks are placed between the TATA box and the transcription initiation site of URA3. Insertion of any sequence between hooks results in inactivation of URA3 expression. That inactivation confers resistance to 5-fluoroorotic acid, allowing selection of TAR cloning events against background vector recircularization events. CONCLUSION: The new system greatly expands the area of application of TAR cloning by allowing isolation of any chromosomal region from eukaryotic and prokaryotic genomes regardless of the presence of autonomously replicating sequences.