Deletion Endpoints Research Articles

Feature Mapping of the HLA Class I Region: Localization of thePOU5F1andTCF19Genes

The class I region of the human leukocyte antigen (HLA) complex located on chromosome 6p21.3 is gene dense. To define the gene content of the class I region, we are constructing genomic DNA feature maps. Here we report mapping of thePOU5F1andTCF19genes to an ∼0.2-Mb region between theHLA-Cand theSgenes. Localization of these genes was facilitated by subcloning genomic DNA fragments from the 0.2-Mb region into a transposon γδ-based vector, selecting transposon-mediated deletionsin vivoinEscherichia coli,and sequencing a nested subset chosen for their uniform distribution of deletion endpoints. ThePOU5F1andTCF19genes are located ∼130 kb telomeric of theHLA-Clocus, approximately 0.6 kb apart from each other. Complete sequencing of a 5.5-kbEcoRI fragment containing theTCF19gene revealed that it is composed of three exons, bounded by consensus splice signals. These experiments illustrate that the transposon-based nested deletion sequencing method provides an easy and efficient approach to feature mapping genomic fragments and to high-resolution analysis of selected subportions.

IS1-mediated intramolecular rearrangements: formation of excised transposon circles and replicative deletions.

A system is described which permits visualization and analysis of a number of molecular species associated with transposition activity of the bacterial insertion sequence, IS1, in vivo. The technique involves induction of an IS1 transposase gene carried by a plasmid which also includes an IS1-based transposable element. It is, in principle, applicable to the identification of transposition intermediates as well as unstable transposition products and those which are not detectable by genetic means. Thirteen novel molecular species were detected after 4 h of induction. Five major species were characterized, based on their behaviour as a function of time, on their hybridization patterns and on the nucleotide sequences of the transposon-backbone junctions. All result from intramolecular IS1 transposition events. The two reciprocal partner products of IS1-mediated deletions, the intramolecular equivalent of co-integrates generated by intermolecular transposition, have been identified. Both carry a single copy of the transposable element and present complementary distributions of deletion endpoints. These results establish, by direct physical means, that adjacent IS1-mediated deletions are accompanied by duplication of the element. A second type of molecule identified was an excised circular copy of the transposon, raising the possibility that IS1 is capable of following an intermolecular transposition pathway, via excised transposon circles, leading to direct insertion.

A new mobile genetic element in Lactobacillus delbrueckii subsp. bulgaricus.

A new IS element (ISL3) was discovered in Lactobacillus delbrueckii subsp. bulgaricus during the characterization of the linkage relationships between the two genes important for milk fermentation, beta-galactosidase (lacZ) and the cell-wall associated protease (prtP). ISL3 is a 1494 bp element, flanked by 38 bp imperfect inverted repeats, and generates an 8 bp target duplication upon insertion. It contains one open reading frame, encoding a potential polypeptide of 434 amino acids, which shows significant homology (34% identity) to the transposase of the Leuconostoc mesenteroides element IS1165. Molecular analysis of spontaneous lacZ mutants revealed some strains that had sustained deletions of 7 to 30 kb in size, centered on and eliminating the copy of ISL3 next to lacZ. Other deletion endpoints were identified as located immediately adjacent to ISL3. Furthermore, genetic translocations that had occurred via transposition of ISL3 were observed fortuitously in cultures screened for deletion mutants. ISL3 can be found in one to several copies in various strains of L. delbrueckii. However, it was not present in other dairy lactic acid bacteria tested.

The Wsh and Ph mutations affect the c-kit expression profile: c-kit misexpression in embryogenesis impairs melanogenesis in Wsh and Ph mutant mice.

The receptor tyrosine kinases (RTKs) c-kit and platelet-derived growth factor receptor alpha chain (PDG-FRa) are encoded at the white spotting (W) and patch (Ph) loci on mouse chromosome 5. While W mutations affect melanogenesis, gametogenesis, and hematopoiesis, the Ph mutation affects melanogenesis and causes early lethality in homozygotes. W-sash (Wsh) is an expression mutation and blocks c-kit expression in certain cell types and enhances c-kit expression in others, including at sites important for early melanogenesis. We have determined the effect of Ph on c-kit expression during embryogenesis in Ph heterozygotes. Immunohistochemical analysis revealed enhanced c-kit expression in several cell types, including sites important for early melanogenesis. We propose that in both Wsh and Ph mutant mice c-kit misexpression affects early melanogenesis and is responsible for the pigment deficiency. Moreover, we have defined the organization of the RTKs in the W/Ph region on chromosome 5 and characterized the Wsh mutation by using pulsed-field gel electrophoresis. Whereas the order of the RTK genes was determined as Pdgfra-c-kit-flk1, analysis of the Wsh mutation revealed that the c-kit and Pdgfra genes are unlinked in Wsh, presumably because of an inversion of a small segment of chromosome 5. The Ph mutation consists of a deletion including Pdgfra and the 3' deletion endpoint of Ph lies between Pdgfra and c-kit. Therefore, positive 5' upstream elements controlling c-kit expression in mast cells and some other cell types are affected by the Wsh mutation and negative elements are affected by both the Wsh and the Ph mutation.

Truncating α-Helix E′ of p66 Human Immunodeficiency Virus Reverse Transcriptase Modulates RNase H Function and Impairs DNA Strand Transfer

The properties of recombinant p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) containing C-terminal truncations in its p66 polypeptide were evaluated. Deletion end points partly or completely removed alpha-helix E' of the RNase H domain (p66 delta 8/p51 and p66 delta 16/p51, respectively), while mutant p66 delta 23/p51 lacked alpha E' and the beta 5'-alpha E' connecting loop. Although dimerization and DNA polymerase properties of all mutants were not significantly different from those of the parental enzyme, p66 delta 16/p51 and p66 delta 23/p51 RT lacked ribonuclease H (RNase H) activity. In contrast, RT mutant p66 delta 8/p51 retained endonuclease activity but lacked the directional processing feature of the parental enzyme. Despite retaining full endoribonuclease function, p66 delta 8/p51 RT barely supported transfer of nascent (-)-strand DNA between RNA templates representing the 5' and 3' ends of retroviral genome, shedding light on the requirement for the endonuclease and directional processing functions of the RNase H domain during replication.

Correlation of doxorubicin footprints with deletion endpoints in lacO of E. coli

This study explored the possibility that the sequence location of doxorubicin-induced deletion endpoints might relate to DNA structural alterations caused by doxorubicin binding to DNA. The 3′-OH endpoints of doxorubicin-induced deletions terminating in the 35-bp region of lacO appear to distribute differently from spontaneous deletion endpoints. Doxorubicin-induced deletions focus in the 26-bp palindrome which is separated by a 9-bp region with no reverse complementary, whereas spontaneous deletion 3′-OH endpoints are found distributed throughout the operator region. In order to explore the mechanism of deletion induction by doxorubicin, drug footprinting studies were carried out with DNA labeled at the 5′ end of each of the complementary DNA strands encompassed by lacO. Doxorubicin protected the 9-bp region between the palindromic sequences from DNase I cutting and caused enhanced DNase I cleavage at symmetrical sites in the palindrome, which were inherently resistant to the nuclease in the absence of the drug. These symmetrical sites also define regions in which the occurrence of deletion endpoints is enhanced 6-fold in the presence of doxorubicin. This enhanced cutting and mutation occur in regions of the palindrome that are flanked by expected doxorubicin binding sites, but are not themselves binding sites of the drug. Similarly, other sites where the frequency of deletion endpoints increased in response to doxorubicin occurred directly adjacent to regions where doxorubicin appeared to inhibit cutting by DNase I. These results suggest that the binding of doxorubicin in the palindrome directs both the frequency and the specificity of deletion formation in this gene region.

Yeast artificial chromosome cloning of the Xq13.3-q21.31 region and fine mapping of a deletion associated with choroideremia and nonspecific mental retardation.

Microscopically detectable deletions and X;autosome translocations have previously facilitated the construction of a high-resolution interval map of the Xq21 region. Here, we have generated three yeast artificial chromosome contigs spanning approximately 7 megabases of the Xq13.3-q21.31 region. In addition, a novel deletion associated with choroideremia and mental retardation was identified and mapped in detail. The proximal deletion endpoint was positioned between the loci DXS995 and DXS232, which enabled us to confirm the critical region for a locus involved in mental retardation. The distal deletion endpoint is situated in the Xq21.33 band, which allowed us to refine the order of several markers in this region.

Mapping the End Points of Large Deletions Affecting the hprt Locus in Human Peripheral Blood Cells and Cell Lines

We have examined the extent of HPRT- total gene deletions in three mutant collections: spontaneous and X-ray-induced deletions in TK6 human B lymphoblasts, and HPRT- deletions arising in vivo in T cells. A set of 13 Xq26 STS markers surrounding hprt and spanning approximately 3.3 Mb was used. Each marker used was observed to be missing in at least one of the hprt deletion mutants analyzed. The largest deletion observed encompassed at least 3 Mb. Nine deletions extended outside of the mapped region in the centromeric direction (> 1.7 Mb). In contrast, only two telomeric deletions extended to marker 342R (1.26 Mb), and both exhibited slowed or limited cell growth. These data suggest the existence of a gene, within the vicinity of 342R, which establishes the telomeric limit of recoverable deletions. Most (25/41) X-ray-induced total gene deletion mutants exhibited marker loss, but only 1/8 of the spontaneous deletions encompassed any Xq26 markers (P = 0.0187). Furthermore, nearly half (3/8) of the spontaneous 3' total deletion breakpoints were within 14 kb of the hprt coding sequence. In contrast, 40/41 X-ray-induced HPRT- total deletions extended beyond this point (P = 0.011). Although the overall representation of total gene deletions in the in vivo spectrum is low, 4/5 encompass Xq26 markers flanking hprt. This pattern differs significantly from spontaneous HPRT- large deletions occurring in vitro (P = 0.032) but resembles the spectrum of X-ray-induced deletions.

Construction of a genomic DNA 'feature map' by sequencing from nested deletions: application to the HLA class I region.

We are applying a transposon-based approach for detecting and mapping features of special interest to construct 'feature maps' of currently uncharacterized portions of the human leukocyte antigen (HLA) complex on chromosome 6. Such feature maps should facilitate identifying regions for high resolution analysis. Here we describe the feature mapping of a 35 kb DNA fragment located between the HLA-C and HLA-E loci. This fragment was cloned into a transposon gamma delta-based cosmid vector designed for generating nested deletions in vivo. Seventy informative nested deletions extending into the cloned fragment were isolated, and DNA adjacent to the deletion endpoints was sequenced by fluorescent automated technology. These islands of DNA sequences constituted the foundation of the feature map, and (i) identified putative exons, (ii) determined the positions of Alu elements, (iii) determined the span of the keratinocyte-specific S gene, and (iv) localized evolutionarily conserved sequences. The construction of feature maps using this in vivo nested deletion-sequencing approach provides a rapid and efficient means to identify DNA regions that merit more detailed analysis.

Filipino beta-thalassemia due to a large deletion: identification of the deletion endpoints and polymerase chain reaction (PCR)-based diagnosis.

The gene frequency of beta-thalassemia among Filipinos is estimated to be 0.02, although little is known about the mutations involved. Recently, an extensive beta-thalassemia deletion was reported in several unrelated individuals of Filipino descent. The deletion begins approximately 4 kb upstream of the beta-globin gene, and extends 3' beyond the beta-globin gene. In this report, we identify the 5' and 3' deletion endpoints and present a polymerase chain reaction (PCR) strategy for rapid DNA diagnosis of the Filipino beta-thalassemia deletion.

Integrons found in different locations have identical 5' ends but variable 3' ends

The positions of the outer boundaries of the 5'- and 3'-conserved segment sequences of integrons found at several different locations have been determined. The position of the 5' end of the 5'-conserved segment is the same for six independently located integrons, In1 (R46), In2 (Tn21), In3 (R388), In4 (Tn1696), In5 (pSCH884), and In0 (pVS1). However, the extent of the 3'-conserved segment differs in each integron. The sequences of In2 and In0 diverge first from the conserved sequence, and their divergence point corresponds to the 3'-conserved segment endpoint defined previously (H.W. Stokes and R.M. Hall, Mol. Microbiol. 3:1669-1683, 1989), which now represents the endpoint of a 359-base deletion in In0 and In2. The sequence identity in In3, In1, In4, and In5 extends beyond this point, but each sequence diverges from the conserved sequence at a different point within a short region. Insertions of IS6100 were identified adjacent to the end of the conserved region in In1 and 123 bases beyond the divergence point of In4. These 123 bases are identical to the sequence found at the mer end of the 11.2-kb insertion in Tn21 but are inverted. In5 and In0 are bounded by the same 25-base inverted repeat that bounds the 11.2-kb insert in Tn21, and this insert now corresponds to In2. However, while In0, In2, and In5 have features characteristic of transposable elements, differences in the structures of these three integrons and the absence of evidence of mobility currently preclude the identification of all of the sequences associated with a functional transposon of this type.

Molecular analysis of aniridia patients for deletions involving the Wilms' tumor gene.

A human aniridia candidate (AN) gene on chromosome 11p13 has been cloned and characterized. The AN gene is the second cloned gene of the contiguous genes syndrome WAGR (Wilms' tumor, aniridia, genitourinary malformations, mental retardation) on chromosome 11p13, WT1 being the first gene cloned. Knowledge about the position of the AN and WT1 genes on the map of 11p13 makes the risk assessment for Wilms' tumor development in AN patients possible. In this study, we analyzed familial and sporadic aniridia patients for deletions in 11p13 by cytogenetic analyses, in situ hybridization, and pulsed field gel electrophoresis (PFGE). Cytogenetically visible deletions were found in 3/11 sporadic AN cases and in one AN/WT patient, and submicroscopic deletions were identified in two sporadic AN/WT patients and in 1/9 AN families. The exact extent of the deletions was determined with PFGE and, as a result, we could delineate the risk for Wilms' tumor development. Future analyses of specific deletion endpoints in individual AN cases with the 11p13 deletion should result in a more precise risk assessment for these patients.

Regulatory sequences involved in the peroxidase gene expression in Arabidopsis thaliana

Organ-specific expression of two peroxidase genes (prxCa and prxEa) from Arabidopsis thaliana was studied. The prxCa gene showed non-specific expression with relatively high levels of mRNA accumulation in the roots, stems and leaves of A. thaliana. The prxEa gene, on the other hand, accumulated high levels of mRNA only in roots. Promoter fragments from each gene were fused to the coding region of β-glucuronidase (gusA) reporter gene introduced into tobacco. Promoter/gusA contructs were transferred to tobacco (Nicotiana tabacum BY-2) protoplasts by electroporation or to N. tabacum SR-1 by Agrobacterium tumefaciens-mediated leaf disk transformation. Transient expression in tobacco protoplasts showed that the 580 fragment from prxEa (Ea-580) expressed thirteen-fold and eight-fold higher GUS activity than prxCa (Ca-622) fragment and CaMV35S promoter, respectively. Tobacco plants transformed with the gusA gene, fused to the -580 deletion (Ea-580), exhibited high GUS expression in roots. The root-specific expression of GUS gene was also observed when the -281 bp deletion end point was used. Although the GUS activity in transgenic tobacco under the control of Ca-622 was low, the activity was found in all organs examined. Histochemical analyses of stem and root tissues of Ea-580 showed that the GUS gene was expressed specifically in phloem and pith parenchyma cells. For Ca-622, high level, specific expression of the gusA gene was observed in the xylem of roots. The results of this study implicate multiple cis-elements in the control of transcription from the prxEa promoter.

Mutation spectrum in the CHM gene of Danish and Swedish choroideremia patients.

The recent isolation of the complete open reading frame of the choroideremia (CHM) gene and the characterization of the exon-intron boundaries has paved the way to mutation detection in patients with classical choroideremia. We have performed mutation screening in patients from 15 Danish and Swedish families by using Southern blot hybridization and the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) technique. Causative mutations in the CHM gene were detected in at least 12 families, indicating that a substantial part of the mutations can be identified by this approach. In four of these families deletions of different sizes were found. Thus, in one patient, the deletion resulted in the absence of only one exon, while in another the deletion comprised the entire CHM gene. Mapping of the deletion endpoints in these four patients and in another 11 male patients with sizeable deletions enabled us to construct a very detailed map of intervals 2 and 3 of Xq21. In the remaining 11 Danish and Swedish families at least 8 causative mutations were found by PCR-SSCP analysis and direct sequencing. Interestingly, all CHM gene mutations detected thus far in choroideremia patients give rise to the introduction of a premature stop codon.

Excision repair reduces doxorubicin-induced genotoxicity

LacI mutations induced by doxorubicin in a wild type, uvr(A)BC repair-proficient E. coli strain were analyzed by DNA sequencing. These mutations were contrasted with mutations previously recovered from doxorubicin-treated vrB− organisms in order to assess the role of excision repair in doxorubicin-induced genotoxicity. After a 30-min exposure of wild-type E. coli to 330 μM doxorubicin, survival was 34% and the overall lacI mutation frequency increased 1.8-fold to 340 × 10−8. The distribution of doxorubicin-induced mutants among subclasses of mutation involving the i−d and lac operator regions differed significantly between repair-proficient and -deficient strains. Distributional differences appeared to result both from a decrease in deletions involving the lac operator and an increase in base substitutions involving the i−d region in repair proficient organisms. However, elements of the doxorubicin-induced mutation spectrum in uvrB−E. coli are still discernable in wild-type organisms. These elements include the remarkable shift of 3′-deletion endpoints to palindromic sequence within the lac operator and the recovery of multiple isolates of T:A → A:T transversions at position 96 in doxorubucin-treated cultures. These observations suggest that components of the uvr(A)BC) nucleotide excision repair system function through a general mechanism prior to fixation of mutations to reduce, but not completely eliminate, the genotoxic effects of doxorubicin.

PDUAL: a transposon-based cosmid cloning vector for generating nested deletions and DNA sequencing templates in vivo.

We describe a transposon gamma delta-containing cosmid cloning vector, pDUAL (previously called pJANUS), and demonstrate an efficient strategy for isolating nested deletions in both large-scale and small-scale DNA sequencing efforts. This "deletion factory" strategy takes advantage of the ability of gamma delta (Tn1000) to generate deletions that extend from an end of the transposon into adjacent DNA when gamma delta transposes to new sites in the same DNA molecule. pDUAL contains the contraselectable (conditional lethal) sacB+ (sucrose sensitivity) and strA+ (streptomycin sensitivity) genes just outside each end of an engineered gamma delta and selectable kan+ (Kanr) and tet+ (Tetr) genes between the cloning site and sacB and strA, respectively. Selection on sucrose tetracycline medium yields deletions that extend from one gamma delta end for various distances into the cloned DNA, while selection on streptomycin kanamycin medium yields comparable deletions in the other direction. Both types of deletions are recoverable because the essential plasmid replication origin is embedded in the gamma delta component and is thereby retained in each deletion product. Pilot experiments with pDUAL clones showed that deletion end points can be mapped or selected by plasmid size and that both DNA strands of any single clone can be accessed for sequencing by using a pair of universal primers specific for sequences that are just interior to the gamma delta ends.

Mutations in POL1 increase the mitotic instability of tandem inverted repeats in Saccharomyces cerevisiae.

Tandem inverted repeats (TIRs or hairpins) of 30 and 80 base-pair unit lengths are unstable mitotically in yeast (Saccharomyces cerevisiae). TIR instability results from deletions that remove part or all of the presumed hairpin structure from the chromosome. At least one deletion endpoint is always at or near the base of the hairpin, and almost all of the repaired junctions occur within short direct sequence repeats of 4 to 9 base pairs. The frequency of this event, which we call "hairpin excision," is influenced by chromosomal position, length of the inverted repeats, and the distance separating the repeat units; increasing the distance between the inverted repeats as little as 25 base pairs increases their chromosomal stability. The frequency of excision is not affected by representative rad mutations, but is influenced by mutations in certain genes affecting DNA synthesis. In particular, mutations in POL1/CDC17, the gene that encodes the large subunit of DNA polymerase I, increase the frequency of hairpin deletions significantly, implicating this protein in the normal maintainance of genomic TIRs.

Precise Deletions in Large Bacterial Genomes by Vector-mediated Excision (VEX): The trfA Gene of Promiscuous Plasmid RK2 is Essential for Replication in Several Gram-negative Hosts

We have developed a simple and efficient method of vector-mediated excision (VEX) for in vivo generation of precisely defined deletion in large bacterial genomes. The system uses homologous recombination with small cloned fragments on specialized pVEX plasmids to insert directly repeated bacteriophage P1 loxP sites at positions flanking the region to be deleted. In the presence of Cre recombinase, the loxP sites are efficiently recombined to produce the deletion. Deletion endpoints can be directed to specific nucleotides because they are defined by the termini of small homology-bearing fragments cloned into the pVEX plasmids. We have used VEX to delete trfA, the only known replication initiator gene of the 56·4 kb broad host-range plasmid RK2. The RK2ΔtrfA mutant was found to be conjugation proficient, but unable to replicate in the RK2 hosts Acinetobacter calcoaceticus, Caulobacter crescentus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas putida, Rhizobium meliloti, or Rhodobacter sphaeroides. These results show that trfA is essential for replication in these hosts and indicate that the broad host-range of RK2 does not involve multiple replicons.

In Planta Deletion of DNA Inserts from the Large Intergenic Region of Cauliflower Mosaic Virus DNA

RNA splicing and copy-choice recombination lead to deletions in the DNA of cauliflower mosaic virus (CaMV). To assess the relative importance of these mechanisms of nucleotide sequence change, free of constraints imposed by the "relay race" mode of translation of CaMV RNA, we examined the stability of inserts in the large intergenic region. The insertions had, in various combinations, splicing signals and directly repeated sequences that could facilitate deletion. Most modified DNAs were infectious. Viral DNA recovered from infected plants was analyzed by restriction and, in some cases, cloned for nucleotide sequencing to determine deletion endpoints. Deletions from a DNA containing introduced splicing signals occurred primarily at direct repeats, although deletion apparently by splicing was also detected. Both types of deletion were also observed with insertions containing a 5′ splice donor but no known functional 3′ splice acceptor. One DNA, whose insertion lacked splicing signals and short repeated sequences, was stable in one of two plants infected. Total insert deletions were bounded by repeats or pseudorepeats, while partial insert deletions apparently occurred by splicing. The results suggest that the two mechanisms for deletion of nucleotides are equally important in the evolution of caulimoviral nucleotide sequences.

Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae.

The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene.