The induction of chromosome aberrations by restriction endonucleases that produce blunt-end or cohesive-end double-strand breaks

Restriction endonucleases have been used to study the involvement of specific types of DNA damages in the production of chromosome aberrations. In this study restriction endonucleases were introduced into viable CHO cells using osmolytic shock of pinocytic vesicles. We compared two cohesive-end cutters, Msp I (CCGG-2-base overlap) and Sau3A I (GATC-4-base overlap) with two blunt-end cutters, Alu I (AGCT) and Rsa I (GTAC). All 4 enzymes were effective at inducing aberrations. The 4-base overlap cohesive-end cutter Sau3A I was approximately as effective as the blunt-end cutter Alu I. We present evidence that cutting frequency rather than cut end-structure is important in determining efficiency of aberration induction. There is over-dispersion of the distribution of dicentrics and rings among cells, and the data could be fitted to a Neyman Type A distribution, a modified Poisson, that indicates that there is a probability distribution both for the entry of the enzyme into a cell nucleus and for the induction of aberrations once the enzyme has entered a cell nucleus. In addition, we used Alu I to determine the sensitivity of cells to aberration induction in the different stages of the cell cycle. Alu I induced aberrations in all stages of the cycle, chromatid-type in S/G2 and chromosome-type in G1. In agreement with data of others, there were variations in sensitivity with cycle stage, and changes in the proportions of the different aberration classes for chromatid-type aberrations.     

The Association Between HLA-A Alleles and Young Alu Dimorphisms Near the HLA-J, -H,and -F Genes in Workshop Cell Lines and Japanese and Australian Populations

At least two polymorphic Alu insertions have been previously identified and characterized within the class I region of the major histocompatibility complex (MHC). We have identified another two new polymorphic Alu insertions, AluyHJ and AluyHF, located near HLA-J and HLA-F, respectively, within the a block of the MHC. Here we report on (1) the haplotypic relationships between the Alu dimorphisms and the HLA-A locus within a panel of 51 IHW homozygous cell lines representing at least 36 HLA class I haplotypes, (2) the Alu genotype, allele, and haplotype frequencies present in the Australian Caucasians and Japanese populations, and (3) the frequency of association between the different Alu dimorphisms and the HLA-A alleles in 109 Australian Caucasians and 99 Japanese. PCR was used to detect the presence or absence of insertion for AluyHJ, AluyHG, and AluyHF within the DNA samples prepared from the cell lines and the two population groups that had been previously typed for HLA-A. In the homozygous cell lines, all three Alu insertions were found in only one HLA class I haplotype (HLA-A1, -B57, -Cw6), no Alu insertions were detected in six HLA class I haplotypes and one or more of the Alu insertions were found in 29 HLA class I haplotypes. At least one of the Alu insertions was found in about 86% of the Japanese and Australian individuals, with the AluyHJ generally related inversely to AluyHG and/or AluyHF. The gene frequency of the AluyHJ and AluyHF insertions was significantly different (p <0.05) BETWEEN JAPANESE AND AUSTRALIANS, WHEREAS THERE WAS NO DIFFERENCE (P > 0.05) between the frequencies of AluyHG in the two populations. The Alu haplotype frequencies were also significantly different between the Japanese and the Australians. In the cell lines and the population groups, the AluyHJ insertion was most frequently found associated with HLA-A1 or A24, AluyHG with HLA-A2, and AluyHF with HLA-A2, -A10, or -A26. This study suggests that the three polymorphic Alu elements have been inserted into the a block of the MHC in different progenitor groups and therefore will be useful lineage and linkage markers in human population studies and for elucidating the evolution of HLA class I haplotypes.     

Elevation of Alu I-induced frequencies of chromosomal aberrations in Chinese hamster ovary cells by Neurospora crassa endonuclease and by ammonium sulfate

The frequencies of chromosomal aberrations induced by the restriction endonuclease Alu I (recognition site AG/CT) can be elevated to a similar extent by additional treatments with a single-strand-specific endonuclease from Neurospora crassa (EC 3.1.30.1), or with ammonium sulfate in which the Neurospora endonuclease is suspended. These data indicate that Alu I does not produce DNA single-strand breaks in the chromatin of living cells, which can be recognized by the Neurospora endonuclease. The salt may induce conformational changes in the chromatin which make more recognition sites available for Alu I. Experiments with recovery times between the treatments with Alu I and the salt indicate that Alu I can act in the nucleus for at least 40 min.     

Evidence for a receptor-mediated endocytosis of Alu I in Chinese hamster ovary cells

Pretreatment of Chinese hamster ovary cells with proteases or with NaN3 leads to less chromosomal aberrations when the cells are posttreated with Alu I compared to the treatment of cells with Alu I alone. The same result is obtained when the cells are treated with Alu I at 0 degree C instead of 37 degrees C. The cells recover from the protease treatment when they are kept in medium before treatment with Alu I. These results are interpreted to mean that Alu I is bound by surface receptors and that the Alu I-receptor complexes are internalized by an energy-dependent endocytotic process.     

A NheI macrorestriction map of the Neisseria meningitidis B1940 genome

Abstract A macrorestriction map of the Neisseria meningitidis strain B1940 genome was constructed by two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. Digestion of the genomic DNA with the restriction endonuclease NHe I revealed 15 fragments between 10 kb and 450 kb. The sum of the fragments and resolution of the linearized chromosome yielded a total genome size of about 2.3 Mbp. By overlapping methylation with the Alu I-methylase six Nhe I recognition sites could be blocked. Fragments were ordered by partial/complete 2D-PFGE of genomic DNA with and without prior Alu I methylation, respectively. All nine Alu I-methylase/ Nhe I and 14 Nhe I restriction sites could be mapped on a single circular chromosome. This map will serve as a useful tool for further genetic analysis of meningococci and exemplifies the power of non-radioactive 2D-PFGE techniques to construct large physical genome maps with a single restriction enzyme.     

Hereditary desmoid disease in a family with a germline Alu I repeat mutation of the APC gene

Two families with autosomal dominantly inherited desmoid tumors have recently been shown to have germline mutations at the 3' end of the APC gene. We subsequently identified an Amish family with autosomal dominantly inherited desmoid tumors. Genetic analysis performed on one family member, a 47-year-old man with multiple desmoid tumors and no colon polyps, revealed a protein truncating mutation in the middle of the APC gene. The truncating mutation is the result of a 337-bp insertion of an Alu I sequence into codon 1526 of the APC gene. The presence of a poly(A) tail at the 3' end of the insertion suggests that the Alu I sequence was inserted by a retrotranspositional event. Germline insertions of Alu I sequences have occasionally been reported to cause other genetic diseases including type I neurofibromatosis, hereditary site-specific breast cancer (BRCA2), and hemophilia B. However, this is the first report of a germline mutation of the APC gene resulting from an Alu I insertion.     

The restriction endonuclease Alu I induces chromosomal aberrations in human peripheral lymphocytes in vitro

The restriction endonuclease Alu I (recognition site AG/CT) produces chromosomal aberrations in isolated human peripheral lymphocytes in vitro. The aberrations are of the chromosome-type when the cells are treated in G1 and of the chromatid-type when the cells are treated in late S, early G2. Additional treatment with ammonium sulphate leads to higher aberration frequencies than treatment with Alu I alone.     

Amplification dynamics of human-specific (HS) Alu family members.

We have investigated the distribution of several recently inserted Alu family members within representatives of diverse human groups. Human population studies using 65 unrelated human DNA samples, as well as a familial study to test inheritance, showed that individual Alu family members could be divided into three groups. The first group consisted of relatively older Alu family members which were monomorphic (homozygous) throughout the population tested (HS C3N1 and C4N6). The second group (HS C4N2, C4N5 and C4N8), apparently inserted into other repetitive regions of the genome, resulting in inconclusive results in the PCR test used. However, it is clear that these particular Alu insertions were present in a majority if not all of the loci tested. The third group was comprised of three dimorphic Alu family members (HS C2N4, C4N4 and TPA 25). Only a single Alu family member (TPA 25) displayed a high degree of dimorphism within the human population. This latter example also showed different allele frequencies in different human groups. The isolation and characterization of additional highly dimorphic Alu family members should provide a useful tool for human population genetics.     

Chromosomal aberrations induced by the restriction endonuclease Alu I in Chinese hamster ovary cells: influence of duration of treatment and potentiation by cytosine arabinoside

Induction of chromosomal aberrations by the restriction endonuclease Alu I in Chinese hamster ovary cells (CHO) has been studied. Treatment of cell pellets with Alu I for a time as short as 1 min was found to induce significant increase in the frequency of chromosomal aberrations. Alu I was found to be effective both in trypsinized cells as well as in cells which were collected with a rubber policeman, indicating that trypsinization of cells is not a prerequisite for the entry of the enzyme into the cells. Treatment of cells with Alu I in the presence of 1-beta-D-arabinosylcytosine (ara C) led to an increase in the induced frequency of aberrations, most probably due to the inhibition of ligation of DNA-strand breaks by ara C.     

Species-specific homogeneity of the primate Alu family of repeated DNA sequences

We have determined the base sequence of several cloned Alu family members from the DNAs of a new world monkey (owl monkey) and a prosimian (galago). The three owl monkey Alu family members reported here belong to a single 300 base pair consensus sequence which closely resembles the human Alu family consensus. The galago Alu family members can best be represented as belonging to either of two related but distinct consensus sequences. One of the two galago Alu family subgroups (Type I) more accurately resembles the human consensus sequence than does the other subgroup (Type II). In this work we compare base sequences of human and galago Type I Alu family members. There are several examples of species-specific differences between the human and Type I galago sequences indicating that the Alu family members are effectively homogenized within a species.     

Genetic recombination in a chromosomal translocation t(2;8)(p11;q24) of a Burkitt's lymphoma cell line, KOBK101

We analyzed a chromosomal translocation, t(2;8)(p11;q24), in a Burkitt's lymphoma cell line, KOBK101. The translocation reciprocally occurred between a site about 150 bp upstream from the J5 segment in the Ig kappa-encoding gene on chromosome 2 and the A-rich end of an Alu repetitive element located far downstream from the c-myc gene on chromosome 8. Short segments of both parental chromosomes were deleted at the rearrangement site. A sequence related to the heptamer recognition signal for the V-J recombination of Ig genes and a topoisomerase I-recognition sequence were detected at the breakpoints. The V-J recombination occurred on both chromosome 2 and the translocated chromosome 2p- at the J3 and J4 segments, respectively. The J region on the translocated chromosomes was mutated, as compared with that on the untranslocated chromosome, while the Alu element and its upstream sequence were conserved. These results suggest the following aspects to the chromosomal translocation of this cell line. A V-J recombination seems to have occurred at the proximal end of the J4 segment first, and then the translocation took place in the region between the J4 and J5 segments. The translocation may have been mediated by the functions of topoisomerase I and the Alu repetitive sequence located at the breakpoint, although the possibility cannot be ruled out that the recombination machinery for Ig gene rearrangements functioned irregularly.     

Association of some potential hormone response elements in human genes with the Alu family repeats.

Short interspersed repeats of the Alu family located in promoters of some human genes contain high-affinity binding sites for thyroid hormone receptor, retinoic acid receptor and estrogen receptor. The standard binding sites for the receptors represent variants of duplicated AGGTCA motif with different spacing and orientation (direct, DR, or inverted, IR), and Alu sequences were found to have functional DR-4, DR-2 or variant IR-3/IR-17 elements. In this study we analyzed distribution and abundance of the elements in a set of human genomic sequences from GenBank and their association with Alu repeats. Our results indicate that a major fraction of potentially active DR-4, DR-2 and variant IR-3/IR-17 elements in the genes is located within Alu repeats. Alu-associated DR-2 elements are conserved in primate evolution. However, very few Alu have potential DR-3 glucocorticoid-response elements. Gel-shift experiments with the probe (AUB) corresponding to the consensus Alu sequence just upstream of the RNA polymerase III promoter B-box and containing duplicated AGGTCA motif indicate that the probe interacts in a sequence-specific manner with human nuclear proteins which bind to standard IR-0, DR-1, DR-4 or DR-5 elements. The AUB sequence was also able to promote thyroid hormone-dependent trans-activation of a reporter gene. The results support the view that Alu retroposons played an important role in evolution of regulation of the primate gene expression by nuclear hormone receptors.     

Alu insertion profiling: array-based methods to detect Alu insertions in the human genome

The analysis of the genetic variability associated to Alu sequences was hampered by the absence of genome-wide methodologies able to efficiently detect new polymorphisms/mutations among these repetitive elements. Here we describe two Alu insertion profiling (AIP) methods based on the hybridization of Alu-flanking genomic fragments on tiling microarrays. Protocols are designed to preferentially detect active Alu subfamilies. We tested AIP methods by analyzing chromosomes 1 and 6 in two genomic samples. In genomic regions covered by array-features, with a sensitivity of 2% (AIP1) -4% (AIP2) and 5% (AIP1) -8% (AIP2) for the old J and S Alu lineages respectively, we obtained a sensitivity of 67% (AIP1) -90% (AIP2) for the young Ya subfamily. Among the loci showing sample-to-sample differences, 5 (AIP1) -8 (AIP2) were associated to known Alu polymorphisms. Moreover, we were able to confirm by PCR and DNA sequencing 4 new intragenic Alu elements, polymorphic in 10 additional individuals.     

The restriction endonuclease Alu I induces chromosomal aberrations and mutations in the hypoxanthine phosphoribosyltransferase locus, but not in the Na+/K+ ATPase locus in V79 hamster cells

The restriction endonuclease Alu I induces chromosomal aberrations and mutations in the hypoxanthine phosphoribosyltransferase (HPRT) locus as measured by 6-thioguanine resistance (TGr) in V79 hamster cells. Alu I does not induce mutations in the Na+/K+ ATPase locus as measured by ouabain resistance (OUAr). The data are interpreted to mean that most if not all Alu I-induced TGr mutations represent chromosomal aberrations.     

伊氏锥虫动基体DNA微环的研究

采用限制性内切酶消化、琼脂糖凝胶电泳及分子杂交技术对８株中国伊氏锥虫动基体ＤＮＡ微环进行了比较研究。结果显示,我国伊氏锥虫株之间的ｋＤＮＡ微环序列具有较高的同源性,仅限制酶ＡｌｕＩ,ＨｉｎｆＩ及ＭｂｌＩ的酶解结果显示少数虫株的ｋＤＮＡ微环存在异源序列。这种异源性可以作为伊氏锥虫种内分类的遗传学标志。     

A comparative study on proteins released from metaphase chromosomes after digestion with restriction endonucleases and deoxyribonuclease I

Extensive digestion of Chinese hamster metaphase chromosomes with Alu I, Hae III and Hinf I released up to 40 distinct chromosomal proteins. Some of the proteins released by Hae III or Hinf I were enriched in the protein moiety liberated by Alu I but several proteins released by Hae III were not released by Alu I digestion. The amount of chromosomal protein released by deoxyribonuclease I (DNase I) was comparable to that liberated by the three restriction enzymes so far tested, while only four abundant protein species were detectable in the protein moiety released by DNase I. Two of them with molecular weights of 58,000 and 50,000 were also released by the three restriction enzymes and are similar in size to those found previously in the core-like structure of histone-depleted chromosomes.     

Linkage disequilibrium among RFLPs at the insulin-receptor locus despite intervening Alu repeat sequences.

Multiple mutations of the insulin receptor (INSR) gene have been identified in individuals with extreme insulin resistance. These mutations have included recombination events between Alu repeat units in the tyrosine kinase-encoding beta-chain region of the gene. To evaluate the influence of Alu and dinucleotide repetitive sequences on recombination events within the insulin receptor gene, I examined the degree of linkage disequilibrium between RFLP pairs spanning the gene. I established 228 independent haplotypes for seven RFLPs (two each for PstI, RsaI, and SstI and one for MspI and 172 independent haplotypes which included an additional RFLP with BglII) from 19 pedigrees. These RFLPs span > 130 kb of this gene, and my colleagues and I previously demonstrated that multiple Alu sequences separate RFLP pairs. Observed haplotype frequencies deviated significantly from those predicted. Pairwise analysis of RFLP showed high levels of linkage disequilibrium among RFLP in the beta-chain region of the insulin receptor, but not between alpha-chain RFLPs and those of the beta-chain. Disequilibrium was present among beta-chain RFLPs, despite separation by one or more Alu repeat sequences. The very strong linkage disequilibrium which was present in sizable regions of the INSR gene despite the presence of both Alu and microsatellite repeats suggested that these regions do not have a major impact on recombinations at this locus.     

A BC200-derived element and Z-DNA as structural markers in annexin I genes: Relevance to Alu evolution and annexin tetrad formation

We have identified two types of structural elements in genomic DNA for annexin I that provide physical evidence of genetic events leading to conserved changes in gene structure. The sequence upstream of the transcribed region in human annexin I contained a rare, Alu-like repetitive element with flanking direct repeats, probably derived from the active BC200 gene via germline retroposition. Nucleotide substitutions in this BC200 insert relative to the 7SL gene and its absence in rodent annexins I identified it as a recent primate pseudogene. Phylogenetic analysis showed that the BC200 gene represents a new clade of primate Alu evolution that branched near the time of appearance of the progenitor to the free left Alu monomer, FLAM-C. Separate analysis identified a Z-DNA motif in pigeon annexin I intron 7 that may represent the vestigial recombination site involved in primordial assembly of the annexin tetrad. These distinct structural features in annexin I genes provide insight into the evolution of Alu repeats and the mechanism of annexin tetrad formation.