Deletion Endpoints Research Articles

Cancer-specific chromosome alterations in the constitutive fragile region FRA3B.

We have sequenced 870 kilobases of the FHIT/FRA3B locus, from FHIT intron 3 to intron 7. The locus is AT rich (61.5%) and Alu poor (6. 2%), and it apparently does not harbor other genes. In a detailed analysis of the 308-kilobase region between FHIT exon 5 and the telomeric end of intron 3, a region known to encompass a human papillomavirus-16 integration site and two clusters of aphidicolin-induced chromosome 3p14.2 breakpoints, we have precisely mapped 10 deletion and translocation endpoints in cancer-derived cell lines relative to positions of specific repetitive elements, regions of high genome flexibility and aphidicolin-induced breakpoints. Conclusions are (i) that aphidicolin-induced breakpoint clusters fall close to high-flexibility sequences, suggesting that these sequences contribute directly to aphidicolin-induced fragility; (ii) that 9 of the 10 FHIT allelic deletions in cancer cell lines resulted in loss of exons, with 7 deletion endpoints near long interspersed nuclear elements or long terminal repeat elements; and (iii) that cancer-specific deletions encompass multiple high-flexibility genomic regions, suggesting that fragile breaks may occur at these regions, whereas repair of the breaks involves homologous pairing of flanking sequences with concomitant deletion of the damaged fragile sequence.

Der(22) Syndrome and Velo-Cardio-Facial Syndrome/DiGeorge Syndrome Share a 1.5-Mb Region of Overlap on Chromosome 22q11

Derivative 22 (der[22]) syndrome is a rare disorder associated with multiple congenital anomalies, including profound mental retardation, preauricular skin tags or pits, and conotruncal heart defects. It can occur in offspring of carriers of the constitutional t(11;22)(q23;q11) translocation, owing to a 3:1 meiotic malsegregation event resulting in partial trisomy of chromosomes 11 and 22. The trisomic region on chromosome 22 overlaps the region hemizygously deleted in another congenital anomaly disorder, velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). Most patients with VCFS/DGS have a similar 3-Mb deletion, whereas some have a nested distal deletion endpoint resulting in a 1.5-Mb deletion, and a few rare patients have unique deletions. To define the interval on 22q11 containing the t(11;22) breakpoint, haplotype analysis and FISH mapping were performed for five patients with der(22) syndrome. Analysis of all the patients was consistent with 3:1 meiotic malsegregation in the t(11;22) carrier parent. FISH-mapping studies showed that the t(11;22) breakpoint occurred in the same interval as the 1.5-Mb distal deletion breakpoint for VCFS. The deletion breakpoint of one VCFS patient with an unbalanced t(18;22) translocation also occurred in the same region. Hamster-human somatic hybrid cell lines from a patient with der(22) syndrome and a patient with VCFS showed that the breakpoints occurred in an interval containing low-copy repeats, distal to RANBP1 and proximal to ZNF74. The presence of low-copy repetitive sequences may confer susceptibility to chromosome rearrangements. A 1.5-Mb region of overlap on 22q11 in both syndromes suggests the presence of dosage-dependent genes in this interval.

Deletion of a region that is a candidate for the difference between the deletion forms of hereditary persistence of fetal hemoglobin and deltabeta-thalassemia affects beta- but not gamma-globin gene expression.

The analysis of a number of cases of beta-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the beta-globin locus has led to the identification of several DNA elements that have been implicated in the switch from human fetal gamma- to adult beta-globin gene expression. We have tested this hypothesis for an element that covers the minimal distance between the thalassemia and HPFH deletions and is thought to be responsible for the difference between a deletion HPFH and deltabeta-thalassemia, located 5' of the delta-globin gene. This element has been deleted from a yeast artificial chromosome (YAC) containing the complete human beta-globin locus. Analysis of this modified YAC in transgenic mice shows that early embryonic expression is unaffected, but in the fetal liver it is subject to position effects. In addition, the efficiency of transcription of the beta-globin gene is decreased, but the developmental silencing of the gamma-globin genes is unaffected by the deletion. These results show that the deleted element is involved in the activation of the beta-globin gene perhaps through the loss of a structural function required for gene activation by long-range interactions.

Molecular nature of mutations induced by a high dose of x-rays in spleen, liver, and brain of thelacZ-transgenic mouse

DNA sequences of 103 spontaneous mutants and 102 X-ray-induced mutants of the lacZ transgene from spleen, liver, and brain of the MutaMouse were examined and compared to elucidate characteristics of radiation-induced mutations in vivo. The radiation-induced mutants were isolated from genomic DNA of each tissue collected at 3.5 days after 200 Gy of whole body irradiation. Base substitution was predominant (80% or more) in nonirradiated tissues, while deletion was prevalent (about 55%) in irradiated tissues. The other types of mutation appeared at similar frequencies in both control and irradiated tissues. The size of the deletions was smaller than 438 nucleotides, with a predominance of one basepair deletions in both control and irradiated tissues. A close look at the nucleotides at the deletion endpoints revealed that many of the radiation-induced deletions did not have repeated sequences at the break point termini, whereas all deletions found in unirradiated tissues showed one or more bases of repeated sequences at the termini. Further, eight complex-type deletion mutations were found only in irradiated tissues. Comparison among the three types irradiated tissue did not reveal any tissue-specificity. The data indicate that the molecular nature of mutations induced in tissues with ionizing radiation is different from that of spontaneous mutations.

Several new bacteriophage T4 genes, mapped by sequencing deletion endpoints between genes 56 (dCTPase) and dda (a DNA-dependent ATPase-helicase) modulate transcription1Published in conjunction with A Wisconsin Gathering Honoring Waclaw Szybalski on the occasion of his 75th year and 20years of Editorship-in-Chief of Gene, 10–11 August 1997, University of Wisconsin, Madison, WI, USA.1

We have analyzed DNA of wild-type T4 and of 13 independent large viable deletions isolated by Homyk and Weil (Virology 61 (1974) 505-523) and by Little (Virology 53 (1973) 47-59), by sequencing, cloning, and expression studies. The deletions can be explained by illegitimate recombination between short (4- to 15-bp) ectopic repeats. In four deletions, adjacent regions are partially homologous, and in at least one of them, the base adjacent to the overlap was deleted during recombination. The sequence 5'-GGGC, which has not been associated with T4 deletions in other map regions, occurs within three repeats, and near the repeats in four more of the 13 deletions. Five previously named genes, 69, soc, mrh, modA, and dda were mapped relative to the deletion endpoints. Nine additional ORFs were found interspersed between them. One of these shares some similarities with mrh (modulates rpoH; Frazier and Mosig, Gene 88 (1990) 7-14), and another one resembles modA (coding for an ADP-ribosyl-transferase that modifies RNA polymerase alpha subunits, Skórko et al., Eur. J. Biochem. 79 (1977) 55-66) respectively. We found that the host's heat shock sigma factor, sigma32, is phosphorylated, and that Mrh protein modulates this phosphorylation. The ORF dda.9 downstream of mrh has a patchy similarity with conserved C-terminal segments (motifs) of sigma32; therefore, we call it srh. Another ORF, dda.2 located between modA and dda, shares sequence similarity with sigma70, and we call it srd. We consider the possibility that Srh and Srd act as decoys for sigma32, or sigma70, respectively. Expression of several of the ORFs from cloned DNA appears to be toxic to the host bacteria. Mutant clones only could be constructed from gene 69 and from modA. Moreover, dda.2 (srd)-containing bacteria grow extremely slowly, and they form filaments in liquid cultures. Clones carrying mrh and srh show less severe filamentation. Our results highlight the importance of 'non-essential' genes for phage development and evolution.

Susceptibility to recombination rearrangements of a chimeric plum pox potyvirus genome after insertion of a foreign gene

Infectious RNA transcripts were generated from a chimeric cDNA clone of the plum pox potyvirus (PPV) genome containing the bacterial β-glucuronidase (GUS) gene inserted between the sequences coding for the P1 and HC proteins. An artificial cleavage site specific for the NIa viral proteinase was engineered between the GUS and HC sequences to produce free GUS and HC proteins. The resulting virus PPVGus↓ was stably maintained during the first round of infection, although plants remained symptomless and virus accumulation was delayed with respect to wild-type infection. PPVGus↓ deleted variants, missing between 645 and 1779 nt, were detected in a subsequent plant passage. PPVGus↓ deletions were confined inside the GUS gene, never affecting the P1 and HC coding regions, in contrast with previous reports of deletions in other potyvirus-based vector, in which deletions frequently reached the HC gene. These results suggest that the N-terminus of the PPV HC protein may be essential for virus viability. Analysis of the deletion endpoints showed short stretches of similarity in donor and acceptor RNAs, as well as oligo A tracts conserved in most junction sites, suggesting that deletions in PPVGus↓ might take place by similarity-assisted recombination events.

Depletion of yeast RNase III blocks correct U2 3' end formation and results in polyadenylated but functional U2 snRNA.

Yeast U2 snRNA is transcribed by RNA polymerase II to generate a single non-polyadenylated transcript. A temperature-sensitive yeast strain carrying a disruption in RNT1, the gene encoding a homolog of RNase III, produces 3'-extended U2 that is polyadenylated. The U2 3'-flanking region contains a putative stem-loop that is recognized and cleaved at two sites by recombinant GST-Rnt1 protein in vitro. Removal of sequences comprising the stem-loop structure blocks cleavage in vitro and mimics the effects of Rnt1 depletion in vivo. Strains carrying a U2 gene lacking the Rnt1 cleavage site produce only polyadenylated U2 snRNA, and yet are not impaired in growth or splicing. The results suggest that eukaryotic RNase III may be a general factor in snRNA processing, and demonstrate that polyadenylation is not incompatible with snRNA function in yeast.

Deletion patterns in Argentine patients with Duchenne and Becker muscular dystrophy

The identification of mutations in Duchenne or Becker muscular dystrophy (DMD/BMD) patients is important for carrier detection in these families. We present the patterns of deletions of the dystrophin gene in Argentine population. DNA from 75 patients with DMD/BMD was analyzed by multiplex peR andl in some casesl cDNNSouthern. Deletions were detected in 24 patients (32%) and were mainly clustered in two areas of the dystrophin gene: the 51 end (exons 3–12) and the central part (exons 44-53). 64% of the deletion endpoints lay in the middle region and 34% in the 51 end of the gene. The most frequent sites for deletion-endpoints were in the introns 47 (73.6%)/ 44 (71%)/ 2 (9%)1 7 (9%) and 12 (7%). Thusl the proportion and di~tribution of deletions in our OMO/BMO patients differ from those reported for other populations. Furthermore/ a higher proportion of deletions was observed in familial cases (40%) than in isolated ones (30%)/ in contrast to previously reported data. The effect of the deletion on the reading frame agree with the phenotype in almost all the patients studied. This study will be useful in prenatal diagnosis and diagnosis of other Argentine DMo patients. [Neural Res 1998; 20: 409–414]

Sequence specificity of illegitimate plasmid recombination in Bacillus subtilis: possible recognition sites for DNA topoisomerase I.

Previous work in our group indicated that structural plasmid instability in Bacillus subtilis is often caused by illegitimate recombination between non-repeated sequences, characterized by a relatively high AT content. Recently we developed a positive selection vector for analysis of plasmid recombination events in B. subtilis which enables measurement of recombination frequencies without interference of selective growth differences of cells carrying wild-type or deleted plasmids. Here we have used this system to further analyse the sequence specificity of illegitimate plasmid recombination events and to assess the role of the host-encoded DNA topoisomerase I enzyme in this process. Several lines of evidence suggest that single-strand DNA nicks introduced by DNA topoisomerase I are a major source of plasmid deletions in pGP100. First, strains overproducing DNA topoisomerase I showed increased levels of plasmid deletion. Second, these deletions occurred predominantly (>90% of the recombinants) between non-repeated DNA sequences, the majority of which resemble potential DNA topoisomerase I target sites. Sequence alignment of 66 deletion end-points confirmed the previously reported high AT content and, most importantly, revealed a highly conserved C residue at position -4 relative to the site of cleavage at both deletion termini. Based on these genetic data we propose the following putative consensus cleavage site for DNA topoisomerase I of B.subtilis: 5'-A/TCATA/TTAA/TA/TA-3'.

Slipped misalignment mechanisms of deletion formation: analysis of deletion endpoints

To gain insight into the mechanisms of deletion formation between tandem repeats, Escherichia coli plasmids were engineered to carry a 101 bp tandem duplication within the tetA gene such that deletion of one of the repeats restores an intact tetA gene and tetracycline resistance to the cell. Four base-pair changes were introduced into one of the tandem repeats to serve as genetic markers. After selection for deletion, individual plasmid products were sequenced to deduce where within the repeat the deletion had occurred. Our analysis shows most deletions are fusions of the two repeats in a single 20 bp interval. This is consistent with the simple replication slip-pair model for deletion formation and suggests that this interval may have unusual features that promote deletion. Dimer replicon products have experienced a sister-chromosome exchange event in addition to deletion and carry two tetA loci: a deleted locus showing a similar distribution of endpoints as seen in the monomer products and an unchanged repeat locus. Seemingly reciprocal dimers are occasionally recovered which carry both a deleted and a triplicated tetA locus. These are not truly reciprocal in that the sequence analysis showed that the deletion and triplication had occurred in separate intervals. Sequence analysis of the dimeric products is consistent with predictions from our sister-strand exchange model where slipped alignment of nascent DNA strands induces deletion formation concomitant with sister-chromosome exchange.

Sequence of the FRA3B common fragile region: implications for the mechanism of FHIT deletion.

The hypothesis that chromosomal fragile sites may be "weak links" that result in hot spots for cancer-specific chromosome rearrangements was supported by the discovery that numerous cancer cell homozygous deletions and a familial translocation map within the FHIT gene, which encompasses the common fragile site, FRA3B. Sequence analysis of 276 kb of the FRA3B/FHIT locus and 22 associated cancer cell deletion endpoints shows that this locus is a frequent target of homologous recombination between long interspersed nuclear element sequences resulting in FHIT gene internal deletions, probably as a result of carcinogen-induced damage at FRA3B fragile sites.

Murine albino-deletion complex: high-resolution microsatellite map and genetically anchored YAC framework map.

The murine albino-deletion complex developed as part of the Oak Ridge specific-locus test covers 6-11 cM of chromosome 7. This complex has proven to be a valuable resource for localizing traits to a small target region for positional cloning. In this study, we mapped the endpoints of deletions in this complex using all of the available Mit simple-sequence length polymorphism (SSLP) markers. Concurrently, this mapping has determined the map order of nearly all of the SSLP markers, most of which were previously unresolved. The SSLP-based deletion map was confirmed and genetic distances were determined using the European Collaborative Interspecific Backcross panel of nearly a thousand mice. The average SSLP marker resolution is 0.3-0.4 cM, comparable to the cloning capacity of yeast artificial chromosomes (YACs). The SSLP markers were then used to construct a genetically anchored YAC framework map that further confirms the deletion map. We find that the largest deleted region distal to Tyr is about two to three times larger than the largest proximal deletion region, and the original C3H/101 regions flanking the deletions (moved to an St2A cch/cch background) are smaller than anticipated, which we suggest may result from increased recombination rates immediately flanking the deleted regions.

A deletion in the PHYD gene of the Arabidopsis Wassilewskija ecotype defines a role for phytochrome D in red/far-red light sensing.

The PHYD gene of the Wassilewskija (Ws) ecotype of Arabidopsis contains a 14-bp deletion (the phyD-1 mutation) beginning at amino acid 29 of the reading frame, resulting in translation termination at a nonsense codon 138 nucleotides downstream of the deletion end point. Immunoblot analyses showed that Ws lacks phyD but contains normal levels of phyA, phyB, and phyC. By backcrossing into the Ws and Landsberg erecta genetic backgrounds, we constructed sibling pairs of PHYD+ and phyD-1 lines and of phyB- PHYD+ and phyB- phyD- lines. Hypocotyl lengths after growth under white or red light increased sequentially in strains that were B+D+, B+D-, B-D+, and B-D-. In the Ws genetic background, an increase in petiole length, a reduction in cotyledon area and in anthocyanin accumulation in seedling stems, a diminished effect of an end-of-day pulse of far-red light on hypocotyl elongation, and a decrease in the number of rosette leaves at the onset of flowering were also seen sequentially in these lines. Thus, phyD, which is approximately 80% identical in amino acid sequence to phyB, acts in conjunction with phyB in regulating many shade avoidance responses. The existence of the apparently naturally occurring phyD-1 mutation indicates that phyD is not essential in some natural environments.

Sequence-specific epigenetic effects of the maternal somatic genome on developmental rearrangements of the zygotic genome in Paramecium primaurelia.

In ciliates, the germ line genome is extensively rearranged during the development of the somatic macronucleus from a mitotic product of the zygotic nucleus. Germ line chromosomes are fragmented in specific regions, and a large number of internal sequence elements are eliminated. It was previously shown that transformation of the vegetative macronucleus of Paramecium primaurelia with a plasmid containing a subtelomeric surface antigen gene can affect the processing of the homologous germ line genomic region during development of a new macronucleus in sexual progeny of transformed clones. The gene and telomere-proximal flanking sequences are deleted from the new macronuclear genome, although the germ line genome remains wild type. Here we show that plasmids containing nonoverlapping segments of the same genomic region are able to induce similar terminal deletions; the locations of deletion end points depend on the particular sequence used. Transformation of the maternal macronucleus with a sequence internal to a macronuclear chromosome also causes the occurrence of internal deletions between short direct repeats composed of alternating thymines and adenines. The epigenetic influence of maternal macronuclear sequences on developmental rearrangements of the zygotic genome thus appears to be both sequence specific and general, suggesting that this trans-nucleus effect is mediated by pairing of homologous sequences.

Deletion formation between the two Salmonella typhimurium flagellin genes encoded on the mini F plasmid: Escherichia coli ssb alleles enhance deletion rates and change hot-spot preference for deletion endpoints.

Deletion formation between the 5'-mostly homologous sequences and between the 3'-homeologous sequences of the two Salmonella typhimurium flagellin genes was examined using plasmid-based deletion-detection systems in various Escherichia coli genetic backgrounds. Deletions in plasmid pLC103 occur between the 5' sequences, but not between the 3' sequences, in both RecA-independent and RecA-dependent ways. Because the former is predominant, deletion formation in a recA background depends on the length of homologous sequences between the two genes. Deletion rates were enhanced 30- to 50-fold by the mismatch repair defects, mutS, mutL and uvrD, and 250-fold by the ssb-3 allele, but the effect of the mismatch defects was canceled by the delta recA allele. Rates of the deletion between the 3' sequences in plasmid pLC107 were enhanced 17- to 130-fold by ssb alleles, but not by other alleles. For deletions in pLC107, 96% of the endpoints in the recA+ background and 88% in delta recA were in the two hot spots of the 60- and 33-nucleotide (nt) homologous sequences, whereas in the ssb-3 background > 50% of the endpoints were in four- to 14-nt direct repeats dispersed in the entire 3' sequences. The deletion formation between the homeologous sequences in RecA-independent but depends on the length of consecutive homologies. The mutant ssb allele lowers this dependency and results in the increase in deletion rates. Roles of mutant SSB are discussed with relation to misalignment in replication slippage.

Chromosomal deletions in Streptomyces griseus that remove the afsA locus

We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA- mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome.

Assignment of fragile site 8E (FRA8E) to human chromosome band 8q24.11 adjacent to the hereditary multiple exostoses 1 gene and two overlapping Langer-Giedion syndrome deletion endpoints.

Assignment of fragile site 8E (FRA8E) to human chromosome band 8q24.11 adjacent to the hereditary multiple exostoses 1 gene and two overlapping Langer-Giedion syndrome deletion endpoints.

Analysis of a hotspot for deletion formation within the intron of the chloroplast trnI gene.

The chloroplast genomes of higher plants encode several tRNA genes that contain highly conserved type II introns. Using primers specific to conserved 5' and 3' regions within the introns of the genes trnA (tRNA-ala) and trnI (tRNA-ile) we have PCR amplified parts of these introns from 36 plant species representing a wide range of plant families. Deletions were found in the introns of both tRNA genes. Fourteen species had detectable deletions within the intron of trnI and four species within the intron of trnA. The occurrence of these deletions among the various plant families suggests that the events leading to the formation of these deletions occurred independently many times during the evolution of higher plants. Analysis of the amplified PCR products from the trnI intron suggests that these independent deletions may not be random but appear to fall into two size classes. Several members of each class were cloned and sequenced and the end points of the deletions were mapped. The 3' ends of all deletions studied terminate within the same short region. The 5' ends of the deletions map to two different regions, giving rise to the two size classes. These two 5' deletion endpoint regions show some sequence similarity. Only two of the identified deletions contain directly repeated sequences at the deletion endpoints, a feature associated with homologous recombination. Our results suggest that within the trnI intron, there are preferred sites or "hotspots" for deletion formation involving a novel imprecise recombination mechanism. The significance of these sequences and possible mechanisms for deletion formation are discussed.

Dynamic mutations in human genes: A review of trinucleotide repeat diseases

Dynamic mutations in human genes result from unstable trinucleotide repeats embedded within the transcribed region. The changeable nature of these mutations from generation to generation is in contrast to the static inheritance of other single-gene mutational events, e.g. point mutations, deletions, insertions and inversions, typically associated with Mendelian inheritance patterns. Intergenerational instability of dynamic mutations within families has provided an explanation for the genetic anticipation, leading to increasing severity or earlier age of onset in successive generations, associated with certain inherited disorders. While models for genomic instability presume that trinucleotide repeat expansion results from disruption of the DNA replication process, experimental evidence has not yet been obtained in support of this contention. Nevertheless, examples of unstable trinucleotide repeats continue to increase, although not all are associated with a specific phenotype. Five disorders resulting from small-scale expansions of CAG repeats within the protein-coding region are known: spinobulbar muscular atrophy, Huntington’s disease, spinocerebellar ataxia type 1, dentatorubral-pallidoluysian atrophy (DRPLA) and Machado-Joseph disease. A sixth disorder, Haw River syndrome, is allelic to DRPLA. Five folate-sensitive chromosomal fragile sites characterized to date, viz. FRAXA, FRAXE, FRAXF, FRA11B and FRA16A, all have large-scale CGG repeat expansion. Two disorders, fragile X syndrome and FRAXE mental retardation, result from instability of CGG repeats in the 5’ untranslated region ofFMR1 andF M R2 genes respectively. FRA11B lies close to chromosome 1 1q deletion endpoints in many Jacobsen syndrome patients and may be related to the deletion event producing partial aneuploidy for 1lq. Expansion of FRAXF and FRA16A has not been associated with a phenotype. Myotonic dystrophy results from a large-scale CTG expansion in the 3’ untranslated region of the myotonin protein kinase gene while Friedreich’s ataxia has recently been found to have a large-scale GAA repeat in the first intron ofX25. This article reviews the characteristics of trinucleotide repeat disorders and summarizes current understanding of the molecular pathophysiology.

A LINE element is present at the site of a 300-kb deletion starting in intron 10 of the PAX6 gene in a case of familial aniridia.

We have identified a 300-kb germline deletion in 11p13 in a family with aniridia but no Wilms' tumor. Cloning and sequencing of the breakpoint revealed that the deletion starts in intron 10 of the PAX6 gene and removes the C-terminal part of the proline-serine-threonine rich domain, leaving both DNA-binding domains intact. The PAX6 gene is joined head-to-head to a LINE-1 (L1) element. The L1 is truncated at the 3' end, removing part of ORF2. Sequencing of the L1 element shows that it does not encode a functional transposase and is therefore probably not an active element. These data suggest that the L1 element is normally present at the site of the distal deletion endpoint in 11p13. No extensive sequence homologies are detected at the deletion junction points; however, the PAX6 gene as well as the L1 element have runs of T nucleotides at this position, indicating that the deletion occurred by nonhomologous recombination. Several consensus recognition sequences for topoisomerase I flank the deletion site in both sequences, suggesting an involvement of this enzyme during the deletion-recombination process.