Genetic analysis of the X-chromosomal region 1E-2A of Drosophila melanogaster

Reversion mutagenesis of three single P elements located in the cytogenetic interval 1E-2A at the tip of the X chromosome of Drosophila melanogaster was used to recover new deletions in this chromosomal region. The deletions obtained include small aberrations within region 2A and larger lesions extending from 2A into 1E and 1B. All three screens also yielded terminal deficiencies. The new deficiencies, together with previously characterized rearrangements, were analyzed for their complementation behaviour with the maternal effect locus fs(1)Nasrat and lethal loci in the region. These analyses provide an overall genetic map of the interval 1E-2A. In addition, the smaller deletions were physically mapped within cloned genomic DNA of the 2A region.     

Large deletions within the first intron in <Emphasis Type="Italic">VRN-1</Emphasis> are associated with spring growth habit in barley and wheat

The broad adaptability of wheat and barley is in part attributable to their flexible growth habit, in that spring forms have recurrently evolved from the ancestral winter growth habit. In diploid wheat and barley growth habit is determined by allelic variation at the VRN-1 and/or VRN-2 loci, whereas in the polyploid wheat species it is determined primarily by allelic variation at VRN-1. Dominant Vrn-A1 alleles for spring growth habit are frequently associated with mutations in the promoter region in diploid wheat and in the A genome of common wheat. However, several dominant Vrn-A1, Vrn-B1, Vrn-D1 (common wheat) and Vrn-H1 (barley) alleles show no polymorphisms in the promoter region relative to their respective recessive alleles. In this study, we sequenced the complete VRN-1 gene from these accessions and found that all of them have large deletions within the first intron, which overlap in a 4-kb region. Furthermore, a 2.8-kb segment within the 4-kb region showed high sequence conservation among the different recessive alleles. PCR markers for these deletions showed that similar deletions were present in all the accessions with known Vrn-B1 and Vrn-D1 alleles, and in 51 hexaploid spring wheat accessions previously shown to have no polymorphisms in the VRN-A1 promoter region. Twenty-four tetraploid wheat accessions had a similar deletion in VRN-A1 intron 1. We hypothesize that the 2.8-kb conserved region includes regulatory elements important for the vernalization requirement. Epistatic interactions between VRN-H2 and the VRN-H1 allele with the intron 1 deletion suggest that the deleted region may include a recognition site for the flowering repression mediated by the product of the VRN-H2 gene of barley.     

Regions associated with repression of the barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) <Emphasis Type="Italic">VERNALIZATION1</Emphasis> gene are not required for cold induction

Activity of the VERNALIZATION1 (VRN1) gene is required for flowering in temperate cereals such as wheat and barley. In varieties that require prolonged exposure to cold to flower (vernalization), VRN1 is expressed at low levels and is induced by vernalization to trigger flowering. In other varieties, deletions or insertions in the first intron of the VRN1 gene are associated with increased VRN1 expression in the absence of cold treatment, reducing or eliminating the requirement for vernalization. To characterize natural variation in VRN1, the first intron of the barley (Hordeum vulgare) VRN1 gene (HvVRN1) was assayed for deletions or insertions in a collection of 1,000 barleys from diverse geographical regions. Ten alleles of HvVRN1 containing deletions or insertions in the first intron were identified, including three alleles that have not been described previously. Different HvVRN1 alleles were associated with differing levels of HvVRN1 expression in non-vernalized plants and with different flowering behaviour. Using overlapping deletions, we delineated regions in the HvVRN1 first intron that are associated with low levels of HvVRN1 expression in non-vernalized plants. Deletion of these intronic regions does not prevent induction of HvVRN1 by cold or the maintenance of increased HvVRN1 expression following cold treatment. We suggest that regions within the first intron of HvVRN1 are required to maintain low levels of HvVRN1 expression prior to winter but act independently of the regulatory mechanisms that mediate induction of HvVRN1 by cold during winter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers 1179825, 1179833, 1179836, 1179858.     

Identification of 54 large deletions/duplications in TSC1 and TSC2 using MLPA, and genotype-phenotype correlations

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by mutations in either of two genes, TSC1 and TSC2. Point mutations and small indels account for most TSC1 and TSC2 mutations. We examined 261 TSC DNA samples (209 small-mutation-negative and 52 unscreened) for large deletion/duplication mutations using multiplex ligation-dependent probe amplification (MLPA) probe sets designed to permit interrogation of all TSC1/2 exons, as well as 15–50 kb of flanking sequence. Large deletion/duplication mutations in TSC1 and TSC2 were identified in 54 patients, of which 50 were in TSC2, and 4 were in TSC1. All but two mutations were deletions. Only 13 deletions were intragenic in TSC2, and one in TSC1, so that 39 (73%) deletions extended beyond the 5′, 3′ or both ends of TSC1 or TSC2. Mutations were identified in 24% of small-mutation-negative and 8% of unscreened samples. Eight of 54 (15%) mutations were mosaic, affecting 34–62% of cells. All intragenic mutations were confirmed by LR-PCR. Genotype/phenotype analysis showed that all (21 of 21) patients with TSC2 deletions extending 3′ into the PKD1 gene had kidney cysts. Breakpoints of intragenic deletions were randomly distributed along the TSC2 sequence, and did not preferentially involve repeat sequence elements. Our own 20-plex probe sets gave more robust performance than the 40-plex probe sets from MRC-Holland. We conclude that large deletions in TSC1 and TSC2 account for about 0.5 and 6% of mutations seen in TSC patients, respectively, and MLPA is a highly sensitive and accurate detection method, including for mosaicism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The switching gene swi6 affects recombination and gene expression in the mating-type region of Schizosaccharomyces pombe

Summary The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h ⁹⁰) strains of Schizosaccharomyces pombe. The MT region of h ⁹⁰ comprises three cassette genes: the expression site mat1: 1 and two silent loci, mat2: 2 and mat3: 3. Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes. These deletions only arise if DNA double-strand breaks are present at mat1: 1, which initiate MT switching. Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2: 2 and mat3: 3; in wild-type strains no recombination occurs in K. swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell. Thus swi6 may have an influence on the general chromatin structure in the MT region.     

The location of untranscribed DNA sequences within ras genes essential for eliciting plant growth suppression

Three heterologous ras DNA-coding sequences and their deletion derivatives were introduced into plant cells to investigate the role of the ras-coding sequences, especially conserved regions, in eliciting growth inhibition. All three ras-coding sequences caused a similar inhibition of plant cell growth, and it was the conserved coding regions which were responsible for this inhibitory effect. The 493 bp conserved region within the v-Ha-ras-coding sequence was studied further, and was shown to be responsible for the inhibitory effect. This region is conserved (over 44%) among the three ras genes studied and encodes a catalytic region of the Ras protein. Small deletions at either the 5 or 3 end of this 493 bp sequence could abolish or dramatically reduce the inhibitory effect. A 36 bp region at the 5 end of the 493 bp region was found to be highly conserved between v-Ha-ras and eight different plant ras or ras-related genes based upon analysis of published sequences. Small deletions affecting this highly conserved 36 bp region completely abolished the inhibitory effect, while deletion of a similar number of base pairs in adjacent regions did not. These results indicate that plant growth inhibition by ras DNA requires small regions at both ends of the 493 bp conserved region.     

The meiotic drive system on maize abnormal chromosome 10 contains few essential genes

In maize, a distal portion of abnormal chromosome 10 (Ab10) causes the meiotic drive of itself as well as many unlinked heterochromatic regions known as knobs. The Ab10 drive system, which encodes trans- as well as cis-acting components, occupies a large region of chromosome 10L equivalent to 3% of the genome. Here we describe five new structural mutations of Ab10 (five deletions and a duplication) that arose from a screen for meiotic drive mutants. The high frequency of breakage events, detected both genetically and cytologically, suggest that the chromosome may be especially unstable. Very large deletions within the drive system are female-transmissible and plants homozygous for deficiencies lacking much of this interval can be grown to maturity. The data suggest that few genes required for normal growth and development lie within the portion of Ab10 responsible for meiotic drive. These and other published data suggest that meiotic drive systems tend to evolve in gene-sparse or otherwise information-poor regions of the genome where they are less likely to negatively affect individual fitness.     

Precise mapping of a locus affecting grain protein content in durum wheat

Grain protein content (GPC) is an important factor in pasta and breadmaking quality, and in human nutrition. It is also an important trait for wheat growers because premium prices are frequently paid for wheat with high GPC. A promising source for alleles to increase GPC was detected on chromosome 6B of Triticum turgidum var. dicoccoides accession FA-15-3 (DIC). Two previous quantitative trait locus (QTL) studies found that the positive effect of DIC-6B was associated to a single locus located between the centromere and the Nor-B2 locus on the short arm of chromosome 6B. Microsatellite markers Xgwm508 and Xgwm193 flanking the QTL region were used in this study to develop 20 new homozygous recombinant substitution lines (RSLs) with crossovers between these markers. These 20 RSLs, plus nine RSLs developed in previous studies were characterized with four new RFLP markers located within this chromosome segment. Grain protein content was determined in three field experiments organized as randomized complete block designs with ten replications each. The QTL peaks for protein content were located in the central region of a 2.7-cM interval between RFLP markers Xcdo365 and Xucw67 in the three experiments. Statistical analyses showed that almost all lines could be classified unequivocally within low- and high- protein groups, facilitating the mapping of this trait as a single Mendelian locus designated Gpc-6B1. The Gpc-6B1 locus was mapped 1.5-cM proximal to Xcdo365 and 1.2-cM distal to Xucw67. These new markers can be used to reduce the size of the DIC chromosome segment selected in marker-assisted selection programs. Markers Nor-B2 and Xucw66 flanking the previous two markers can be used to select against the DIC segment and reduce the linkage drag during the transfer of Gpc-6B1 into commercial bread and pasta wheat varieties. The precise mapping of the high GPC gene, the high frequency of recombinants recovered in the targeted region, and the recent development of a tetraploid BAC library including the Gpc-6B1 DIC allele are the first steps towards the map-based cloning of this gene.Communicated by J. Dvorak     

Cytogenetical analysis of the 2B3-4-2B11 region of the X chromosome of Drosophila melanogaster

Summary Of 13 ecs mutations, which affect female fertility, as revealed by complementation analysis, 7 are chromosome rearrangements involving the br complementation group. The other six show no cytologically detectable rearrangements and behave as completely or partially noncomplementing ecs alleles. All viable combinations of these 13 mutations were characterized by partial or complete female sterility. Viable heterozygotes carrying any of these mutations and the rearrangements Df(1)sta, T(1,3)sta, Df(1)St490, previously localized distal to the ecs locus, were also sterile. Using deletions and an electrophoretic mobility variant from the Staket strain, a minor chorion gene S70 has been mapped. It had been thought this gene was located in the 2B3-5 region, and corresponded to the ecs locus. However, in the present study, this gene was shown to map in the region removed by Df(1)sta (1E1-2-2B3-4) but outside that removed by Df(1)At127 (1E1-2-2A1-2), i.e. within the 2A1-2-2B3-4 region which is distal to the ecs locus. Rearrangements and point mutations at the ecs locus that result in female sterility had no effect on synthesis of the chorion protein s70. It may therefore be suggested that the chorion protein gene is not functionally associated with the ecs locus and that sterility is caused not by disruptions of the chorion protein gene but by lesions in the ecs gene itself. Thus, an ecs product, which controlls cell sensitivity to ecdysterone is also necessary for female fertility. Data on the locations of lesions affecting female fertility indicate that at least two elements at the ecs locus are essential for this function: a cis-acting distal zone with no effect on viability and a sequence within the essential part of the ecs locus. A defect in either of these zones or their separation by chromosomal rearrangement leads to female sterility.     

Cytogenetic analysis of chromosome region 89A of Drosophila melanogaster: isolation of deficiencies and mapping of Po, Aldox-1 and transposon insertions

Summary We have initiated a cytogenetic analysis of chromosome region 89A of Drosophila melanogaster by isolating a set of radiation-induced mutations causing loss of function of P[(w)B]1-1, a transposon bearing the white locus inserted in 89A. Complementation tests and cytological examination of these chromosomes identified four new deficiencies (Df(3R)Po ², Df(3R)Po ³, Df(3R)Po ⁴ and Df(3R)c(3)G ² ). The new deficiencies and three previously identified deficiencies (Df(3R)sbd ²⁶, Df(3R)sbd ⁴⁵ and Df(3R)sbd ¹⁰⁵) were tested for the ability to complement mutations in the enzyme loci Po and Aldox-1, the indirect flight muscle genes Tm2 and act88F, the morphological mutations jvl, sbd ² and Sb, the vital loci srp, pnr and mor, and a newly described vital locus l(3)89Aa. We also used linkage analysis to determine the order and relative positions of P[(w)B]1-1 and an independent transposon insertion, P[w⁺]21, with respect to cv-c, Po, Aldox-1 and sbd ². Cytological examination of the deficiencies and analysis of the transformed lines by in situ hybridization permits the correlation of genetically defined regions with specific polytene chromosome bands. A revised cytogenetic map of the 8817–8913 region is presented.     

Deletion of theAlu-VpA/MycL1 (1p34.3) locus is a negative prognostic sign in human colorectal cancer

We examined deletions of the short arm of chromosome 1 and aberrations of the microsatellite locusAlu-VpA/MycL1 (1p34.3) in human primary colorectal adenocarcinomas. Cytogenetically discernible deletions in 1p were found in 45% (14/31) of informative tumors. The 1p-tumors commonly exhibited a polyploid karyotype (FisherP ₁=0.023) and a larger number of rearranged chromosomes (P ₂=0.045) versus those without 1p deletions. The 1p deletions often combined with chromosome 5 monosomy (χ²=6.24; p=0.013), chromosome 15 monosomy (χ²=4.20;p=0.040), and 11q deletions (P ₂=0.035). Among the 50 carcinomas, 11 (22%) showedAlu-VpA/MycL1 instability, and 14% (6/43 informative) had lost theAlu-VpA/MycL1 allele. The genetic alterations thus revealed were collated with the clinical and morphological features of the tumors. The loss of the 1p material was shown to be correlated with marked karyotype aberrations in colorectal tumors, andAlu-VpA/MycL1 allele deletions were tightly associated with relapses or metastasis within 30 months after surgery.     

Functional analysis of a large non-conserved region of FlgK (HAP1) from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The single subpolar flagellum of Rhodobacter sphaeroides shows an enlarged hook-filament junction. One of the two proteins that compose this section of the filament is HAP1 _Rs (FlgK _Rs ) it contains a central non-conserved region of 860 amino acids that makes this protein about three times larger than its homologue in Salmonella enterica serovar Typhimurium. We investigated the role of this central portion of the unusually large HAP1 protein of R. sphaeroides by monitoring the effects of serial deletions in flgK _Rs , the gene encoding HAP1 _Rs , on swimming and swarming. Two deletion mutants did not assemble functional flagella, two were paralyzed and five exhibited reduced free-swimming speeds. Some mutants produced unusual swarming patterns on soft agar without or with Ficoll 400. A segment of approximately 200-aa of the central region of HAP1 _Rs that aligns with the variable region of the flagellin sequence from other γ- and β-proteobacteria was also found. Therefore, it is possible that the origin of this large central domain of HAP1 _Rs could be associated with an event of horizontal transfer and subsequent duplications and/or insertions.     

The agarase gene (dagA) of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis

Summary The oligopeptide permease is encoded by at least four genes which are transcribed as a single operon. We cloned and characterized this operon from Salmonella typhimurium, as well as the flanking genes, tonB, ana and a new gene, cwd, which affects cell wall synthesis. We correlated the physical map of opp DNA with a detailed genetic map of the opp operon and the individual opp genes were accurately located with respect to various restriction sites by Southern blotting. The region of the chromosome near opp was found to be highly unstable with deletions arising at a high frequency. The operon also contains hot-spots for IS1 and IS5 insertions.     

The determination of sense organs in Drosophila: interaction of scute with daughterless

Summary Several genetic loci have been implicated in the formation of the peripheral nervous system during Drosophila embryogenesis. As a first step towards understanding the functional interrelationships between these genes, we have searched for dominant interactions between deficiencies for the achaete-scute complex (AS-C), daughterless (da) and six other regions necessary for peripheral neurogenesis in the embryo. We have found that adult flies doubly heterozygous for deletions of AS-C and of da, or of AS-C and a small region on the fourth chromosome, exhibit characteristic bristle defects, suggesting that these genes cooperate to form sense organs both in the embryo and in the adult.     

Reactivation of the ATCase domain of the URA2 gene complex: a positive selection method for Ty insertions and chromosomal rearrangements in Saccharomyces cerevisiae

Genetic rearrangements such as deletions or duplications of DNA sequences are rarely detected in the yeast Saccharomyces cerevisiae. We have developed a screening system using the URA2 gene coding for the bi-functional CPSase-ATCase (carbamyl phosphate synthetase — aspartate transcarbamylase) to select positively for these kinds of events. Nonsense mutations in the CPSase region cause a complete loss of the ATCase activity because of their strong polar effect. Thirty-seven ATCase⁺ revertants were isolated from a strain containing three nonsense mutations in the proximal CPSase region. Genetic and structural analysis of the URA2 locus in these strains allowed us to characterize two major classes of revertants. In the first, an entire copy of a Ty transposon was found to be inserted in the CPSase coding domain. This event, which represents a new form of Ty-mediated gene activation was further analysed by mapping the Ty integration site in 26 strains. In a second class of revertants, we observed chromosomal rearrangements and, in particular, duplication of the ATCase region and its integration in a new chromosomal environment in which this sequence becomes active.     

High-resolution array comparative genomic hybridization analysis reveals unanticipated complexity of genetic deficiencies on chromosome V in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Genomic rearrangements are widely used in Caenorhabditis elegans research but many remain incompletely characterized at the physical level. We have used oligo-array comparative genomic analysis to assess the physical structure of 20 deficiencies and a single duplication of chromosome V. We find that while deletions internal to the chromosome appear simple in structure, terminal deletions are complex, containing duplications in addition to the deletion. Additionally, we confirm that transposon-induced deficiencies contain breakpoints that initiate at Tc1 elements. Finally, 13 of these deficiencies are known to suppress recombination far beyond the extent of the deletion. These deficiencies fall into two classes: strong and weak suppressors of adjacent recombination. Analysis of the deleted regions in these deficiencies reveals no common physical sites to explain the observed differences in recombination suppression. However, we find a strong correlation between the size of the rearranged chromosome and the severity of recombination suppression. Rearranged chromosomes that have a minor effect on recombination fall within 2% of normal chromosome size. Our observations highlight the use of array-based approaches for the analysis of rearranged genomes, revealing previously unidentified deficiency characteristics and addressing biologically relevant questions.     

Evolution of the transposable element Uhu in five species of Hawaiian Drosophila

The complete DNA sequence of three independent isolates of Uhu, a member of the Tc1-like class of transposable elements from D. heteroneura (Uhu-1, Uhu-3, and Uhu-4), has been determined. These isolates have between 95 and 96.4% nucleotide sequence identity indicating that Uhu is well conserved within this species. A comparison of the DNA sequences of Uhu and the D. melanogaster Hb1 transposable element shows that the nucleotide substitution rate for Uhu is comparable to the synonymous rate for the Adh gene in these species. Uhu has been identified in four other species of endemic Hawaiian Drosophila, D. silvestris, D. differens, D. planitibia and D. picticornis, and nine Uhu elements were isolated from genomic libraries of these four species. A 444 base pair region from within the coding region of the Uhu element, with well conserved ends, was amplified by the polymerase chain reaction and used for sequence comparison of elements from different species. The analysis of the sequence similarities between the elements within and between the species shows a grouping of the two pairs of most closely related species (D. heteroneura and D. silvestris, and D. differens and D. planitibia), but shows a much larger variation within the most recently diverged species (D. heteroneura and D. silvestris) than expected. There are extensive nucleotide substitutions and deletions in the Uhu elements from D. picticornis showing that they are degenerating and being lost in this species. These observations indicate that the Uhu element has been transmitted vertically and that transposition may have been activated at the time of formation of each species as it colonized the newly formed islands of the Hawaiian archipelago.     

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression

Summary Nearly isogenic lines (NILs) of rice (Oryza sativa) differing at a locus conferring resistance to the pathogen Xanthomonas oryzae pv. oryzae were surveyed with 123 DNA markers and 985 random primers using restriction fragment length plymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. One chromosome 11 marker (RG103) detected polymorphism between the NILs that cosegregated with Xa21. All other chromosome 11 DNA markers tested were monomorphic between the NILs, localizing the Xa21 introgressed region to an 8.3 cM interval on chromosome 11. Furthermore, we identified two polymerase chain reaction (PCR) products (RAPD2148 and RAPD818) that detected polymorphisms between the NILs. Genomic sequences hybridizing with RAPD818, RAPD248 and RG103 were duplicated specifically in the Xa21 NIL. All three markers cosegregated with the resistance locus, Xa21, in a F₂ population of 386 progeny. Based on the frequency with which we recovered polymorphic Xa21-linked markers, we estimated the physical size of the introgressed region to be approximately 800 kb. This estimation was supported by physical mapping (using pulsed field gel electrophoresis) of the sequences hybridizing with the three Xa21-linked DNA markers. The results showed that the three Xa21-linked markers are physically close to each other, with one copy of the RAPD818 sequences located within 60 kb of RAPD248 and the other copy within 270 kb of RG103. None of the enzymes tested generated a DNA fragment that hybridized with all three of the markers indicating that the introgressed region containing the resistance locus Xa21 is probably larger than 270 kb.     

Characterization of the gene products produced in minicells by pSM1, a derivative of R100

Summary At least ten polypeptides larger than 6 kilodaltons (K) are produced in minicells from the miniplasmid pSM1 in vivo. pSM1 (5804 bp) is a small derivative of the drug resistance plasmid R100 (ca. 90 kb) and carries the R100 essential replication region as well as some non-essential functions. Cloned restriction fragments of pSM1 and plasmids with deletions within pSM1 sequences were used to assign eight of the ten oberserved polypeptides to specific coding regions of pSM1. Two of these polypeptides were identified as RepA1 and RepA2, proteins encoded by the essential replication region of pSM1/R100. The nucleotide sequence consisting of 885 bp outside the essential replication region is presented here. This sequence contains an open reading frame,orf4, for a protein 22.9 K in size, and one of the pSM1-encoded polypeptides was identified as theorf4 gene product. Five additional polypeptides were shown to be the products of other open reading frames mapping outside the essential replication region. Specific functions have been assigned to four of these polypeptides and tentatively to the fifth.