The structure of the chloroplast genome in members of the genus Asparagus

 In a previous study we constructed a physical map of the chloroplast DNA (ctDNA) of garden asparagus (Asparagus officinalis L. cv ‘Mary Washington 500W’; Lee et al. 1996). In the present study we have constructed and compared HindIII and XhoI restriction maps of the ctDNAs of eight species of Asparagus: namely, A. officinalis, A. schoberioides, A. cochinchinensis, A. plumosus, A. falcatus, A. sprengeri, A. virgatus and A. asparagoides. The ctDNA of A. officinalis has 32 and 23 sites that are recognized by HindIII and XhoI, respectively. Taking the physical map of the ctDNA of A. officinalis as a standard, we found that the ctDNAs of A. falcatus, A. sprengeri, and A. asparagoides each had one additional HindIII site and lacked one XhoI site. We also detected two relatively large deletions of nucleotides in the ctDNA from A. cochinchinensis by sequencing analysis. Both of these deletions were located in a non-coding region between the ndhC and trnV genes and were 95 bp and 347 bp in length, respectively. The regions around the deletions exhibited strong homology, and short direct-repeat sequences were detected at the borders of the deletions, an indication that these deletions were the result of intramolecular recombination mediated by the direct repeats. Received: 16 June 1997 / Accepted: 17 July 1997     

Isolation and sequence analysis ofCaenorhabditis briggsae repetitive elements related to theCaenorhabditis elegans transposon Tc1

Summary We have identified two repetitive element families in the genome of the nematodeCaenorhabditis briggsae with extensive sequence identity to theCaenorhabditis elegans transposable element Tc1. Five members each of the TCb1 (previously known as Barney) and TCb2 families were isolated by hybridization to a Tc1 probe. Tc1-hybridizing repetitive elements were grouped into either the TCb1 or TCb2 family based on cross-hybridization intensities among theC. briggsae elements. The genomic copy number of the TCb1 family is 15 and the TCb2 family copy number is 33 in theC. briggsae strain G16. The two transposable element families show numerous genomic hybridization pattern differences between twoC. briggsae strains, suggestive of transpositional activity. Two members of the TCb1 family, TCb1#5 and TCb1#10, were sequenced. Each of these two elements had suffered an independent single large deletion. TCb1#5 had a 627-bp internal deletion and TCb1#10 had lost 316 bp of one end. The two sequenced TCb1 elements were highly conserved over the sequences they shared. A 1616-bp composite TCb1 element was constructed from TCb1#5 and TCb1#10. The composite TCb1 element has 80-bp terminal inverted repeats with three nucleotide mismatches and two open reading frames (ORFs) on opposite strands. TCb1 and the 1610-bp Tc1 share 58% overall nucleotide sequence identity, and the greatest similarity occurs in their ORF1 and inverted repeat termini.     

Identification of a cis-regulatory region of a gene in Arabidopsis thaliana whose induction by dehydration is mediated by abscisic acid and requires protein synthesis

The effect of the ATP-dependent exonuclease AddAB complex on the structural stability of plasmid pGP1 inBacillus subtilis was studied. Using deletion mutagenesis and gene amplification techniques,B. subtilis strains were constructed either lacking or overproducing the AddAB complex, a key enzyme in homologous recombination. The deletion mutant possessed no residual ATP-dependent nuclease activity; in contrast, the nuclease activity was up to 30 times higher in lysates of strains carrying multiple copies of theaddAB genes in the chromosome. Southern blot analyses of these strains indicated that a linear relationship exists between the number of chromosomal gene copies and the level of AddAB activity. The structural stability of pGP1 was analyzed in the AddAB-deficient and over-producing backgrounds. Frequencies of deletion formation in the plasmid, as monitored by the expression of the pGP1-encodedpenP-lacZ fusion on media containing X-gal, were shown to be increased at least 25-fold in theaddAB knock-out mutant, whereas the stability of pGP1 was improved up to 15-fold in strains verproducing the AddAB enzyme. A possible explanation for these findings is that interactions between AddAB and plasmid molecules prevent the formation of secondary structures that constitute potential deletion target sites, and thereby enhance the structural stability of plasmids.     

A phylogenetic analysis of cattle DRB3 alleles with a deletion of codon 65

 One of the most common cattle major histocompatibility complex DRB3 alleles, ^* 0201, includes a deletion of codon 65 encoding one residue in the α-helical chain. The mutation is functionally interesting and is likely to influence peptide binding. Exon 2 of two additional del65 alleles, ^* 3301 and ^* 4101, have now been sequenced with the aim to investigate the evolutionary relationship of this allelic group. Despite a fairly large genetic distance between the three alleles (11–17 nucleotide substitutions causing 8–11 amino acid substitutions) we found clear indications of a common ancestry. The α-helical region was very similar or identical among the alleles whereas the β-strand region was quite divergent. The results indicated that interallelic recombination has contributed to the diversification of the del65 group. Deletion of codon 65 has also been found in a roe deer DRB1 allele and a cattle DQB3 allele. Sequence comparisons of the cattle and roe deer DRB del65 alleles refuted the possibility of a trans-species persistence of a del65 allelic lineage but the two species may share a short ancestral sequence motif including del65. In addition to del65, the cattle DQB3 allele did not show any striking sequence similarities to the DRB alleles. Received: 20 March 1997 / Revised: 17 June 1997     

通过基因组相减从悬铃木突变体中分离缺失DNA

悬铃木（Ｐｌａｔａｎｕｓ ｏｃｃｉｄｅｎｔａｌｉｓ Ｌ．）突变体是其种子经γ射线辐射培育出的一种营养生长正常而开花发育过程受阻的不育植株。经过１０多年的观察研究证明：突变体的花芽不能形成是因为突变体细胞染色体有部分缺失。应用基因组相减技术分离这些在突变体中缺失但在野生型中存在的ＤＮＡ,经过４次循环富集缺失片段后克隆了１个２．０ ｋｂ 的ＤＮＡ 片段,ＤＮＡ 杂交证明此片段只存在于野生型基因组中而不存在于突变体中。将基因组相减方法应用在复杂植物基因组中获得成功的研究还未见报道     

Heterogeneous genomic amplification in Streptomyces glaucescens: structure, location and junction sequence analysis

Summary Certain chromosomal markers inStreptomyces glaucescens behave unstably, being lost at high frequency as a result of extensive genomic deletion. Additionally, mutant strains possessing such deletions frequently display intense DNA amplification. With the help of a wild-type cosmid library we investigated the structure of the amplified DNA sequences (ADS) and the corresponding wild-type amplifiable units of DNA (AUD). The reiterations were heterogeneous in location, copy number and sequences involved and originated predominantly from a single 100 kb region of the chromosome called the AUD locus. All strains bearing reiterations possessed associated deletions which terminated either close to or at the ADS. The termini of four AUDs were sequenced in order to gain more knowledge about these heterogeneous amplifications. In three of the four cases investigated small, interrupted homologies were found bordering the AUDs. With the help of orthogonal-field-alternation gel electrophoresis (OFAGE) we were able to visualize a tandem reiteration of more than 1500 kb in length.     

Extremely large chromosomal deletions are intimately involved in genetic instability and genomic rearrangements in Streptomyces glaucescens

Summary Genetic instability inStreptomyces glaucescens characteristically involves the occurrence of gross genomic rearrangements including high-level sequence amplification and extensive deletion. We investigated the relationship of the unstablemelC andstrS loci and a 100 kb region of the chromosome which frequently gives rise to intense heterogeneous DNA amplification. Standard chromosome walking using a cosmid bank in conjunction with a “reverse-blot” procedure enabled us to construct a contiguous genomicBamHI map of the unstable region exceeding 900 kb. The unstable genes and the amplifiable region (AUD locus) are physically linked within a 600 kb segment of the chromosome. The previously characterized deletions which affect these loci are merely components of much larger deletions ranging from 270 to over 800 kb which are polar in nature, effecting the sequential loss of thestrS andmelC loci. The more extensive deletions terminate either adjacent to, or in the vicinity of DNA reiterations at the AUD locus. Additionally, a deletion junction fragment and the corresponding deletion ends were cloned and analysed at the sequence level.     

Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria

Summary The avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria was cloned and found to be localized on a self-transmissable plasmid. Genetic analysis of an avrBs3 insertion mutation revealed that avrBs3 constitutes a single locus, specifying the resistant phenotype on pepper plants. Southern blot experiments showed that no DNA sequences homologous to avrBs3 were present in other races of X. c. pv. vesicatoria, which are unable to induce a hypersensitive reaction on ECW-30R. However, the DNA of several different pathovars of X. campestris hybridized to the avrBs3 probe. A deletion analysis defined a region of 3.6–3.7 kb essential for avrBs3 activity. The nucleotide sequence of this region was determined. A 3561 nucleotide open reading frame (ORF1), encoding a 125000 dalton protein, was found in the 3.7 kb region that was sufficient for avrBs3 activity. A second long ORF (2351 nucleotides) was identified on the other strand. A remarkable feature of both ORFs is the presence of 17 direct repeats of 102 bp which share 91%–100% homology with each other.