Molecular cloning of the c2 locus of Zea mays, the gene coding for chalcone synthase

Summary The c2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable element En (Spm) as a gene tag. The Spm element present at the c2 locus in the autonomously mutating c2-m1 line was isolated using En1 element specific probes. Sequences flanking the element were identified as c2 locus specific and were used to clone the nonautonomous c2-m2 and wild-type alleles. The cloning and analysis of a cDNA complementary to the c2 locus provided evidence that this gene encodes the enzyme chalcone synthase.     

Identification and cloning of a hemin storage locus involved in the pigmentation phenotype of Yersinia pestis.

The temperature-dependent absorption of sufficient exogenous hemin or Congo red to form pigmented colonies of Yersinia pestis has been termed the pigmentation phenotype (Pgm+). Spontaneous mutation to a Pgm- phenotype results in the loss of a number of divergent physiological characteristics, including the ability to store hemin and to bind Congo red at 26 degrees C. In this study, we generated and isolated transposon insertion mutants that are hemin storage negative (Hms-) and therefore unable to form pigmented colonies. These mutations are due to single mini-kan insertions within a 19.5-kilobase (kb) SalI fragment of chromosomal DNA. Restriction site analysis of eight mutants identified a minimum of six potentially different insertion sites spanning an approximately 10-kb hemin storage (hms) locus. The 19.5-kb SalI fragment (containing approximately 18 kb of Y. pestis DNA and the mini-kan insert) was cloned from one of these mutants, KIM6-2012. By using this cloned fragment as a DNA probe, the mechanism of spontaneous mutation to a Pgm- phenotype was identified as a massive deletion event. The deletion spans at least 18 kb of genomic DNA in spontaneous Pgm- mutants from nine separate strains of Y. pestis. DNA adjacent to the mini-kan insert was used to identify a clone containing a wild-type hms locus. A spontaneous Pgm- mutant of Y pestis KIM containing this clone exhibits an Hms+ phenotype. The hms::mini-kan mutations and cloned wild-type hms locus generated in this study will greatly aid in identifying the function of hemin storage in Y. pestis.     

Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae

In order to clone and analyse the avirulence gene AVR-Pia from Japanese field isolates of Magnaporthe oryzae , a mutant of the M. oryzae strain Ina168 was isolated. This mutant, which was named Ina168m95-1, gained virulence towards the rice cultivar Aichi-asahi, which contains the resistance gene Pia. A DNA fragment (named PM01) that was deleted in the mutant and that co-segregated with avirulence towards Aichi-asahi was isolated. Three cosmid clones that included the regions that flanked PM01 were isolated from a genomic DNA library. One of these clones (46F3) complemented the mutant phenotype, which indicated clearly that this clone contained the avirulence gene AVR-Pia . Clone 46F3 contained insertions of transposable elements. The 46F3 insert was divided into fragments I–VI, and these were cloned individually into a hygromycin-resistant vector for the transformation of the mutant Ina168m95-1. An inoculation assay of the transformants revealed that fragment V (3.5 kb) contained AVR-Pia . By deletion analysis of fragment V, AVR-Pia was localized to an 1199-bp DNA fragment, which included a 255-bp open reading frame with weak homology to a bacterial cytochrome- c -like protein. Restriction fragment length polymorphism analysis of this region revealed that this DNA sequence co-segregated with the AVR-Pia locus in a genetic map that was constructed using Chinese isolates.     

Cloning Knotted, the dominant morphological mutant in maize using Ds2 as a transposon tag

The Kn1-2F11 mutation causes protrusions or knots along the lateral veins of the first few leaves of the maize plant. The phenotype is visible when an unlinked gene, presumably Ac, is present in the genome. The mutation is closely linked to a genetically unstable Adh1 mutation that resulted from the insertion of a Ds2 element (Döring et al., 1984; Chen et al., 1986). Using a unique sequence from the Ds2 element as a hybridization probe, a genomic restriction fragment that cosegregated with the knotted phenotype was cloned. It carries the Kn1-2F11 locus by the following criteria. (i) Cosegregation of the fragment is tightly linked to the phenotype. (ii) Somatic and germinal excision produce a fragment which is the expected size of a revertant fragment; progeny containing the revertant size fragment are normal. (iii) The sequences that hybridize to this fragment are significantly altered in the chromosome containing the original knotted mutation, Kn1-O, (iv) The cloned fragment does not hybridize to a chromosome that contains a deletion of Kn1-O.     

Cloning of the Bz2 locus of Zea mays using the transposable element Ds as a gene tag

Summary The Bz2 locus of Zea mays has been cloned, utilizing the presence of the transposable element Dissociation (Ds) at the locus as a gene tag. The Ds element inserted in the bz2-m allele was identified among many members of the Ac/Ds family in a Southern blot analysis of a population segregating for bz2-m and Bz2. After cloning a DNA fragment from the bz2-m allele, sequences flanking the Ds insertion were shown to be Bz2-specific and were used to isolate a homologous fragment from a wild-type Bz2 line. The Ds insertion in the bz2-m allele was found to be a Ds2 element identical to the Ds insertion in adh1-2F11.     

不同启动子控制下Ac转座酶基因的表达对玉米Ds因子在水稻中切离频率的影响

用水稻愈伤组织比较了Ac启动子、35S启动子与Ubi启动子控制下Ac转座酶基因（Ts）的表达对Ds因子切离频率的影响。结果表明Ubi启动子与Ac转座酶编码区嵌合基因（Ubipro-Ts）反式激活Ds因子的切离频率最高,达到了72.9%。通过杂交将Ubipro-Ts基因导入Ds因子转化植株,得到9株Ubipro-Ts基因与Ds因子共存的F1代杂交水稻植株,其中有8株Ds因子发生了切离。用Inverse-PCR的方法从其中一株杂交植株中克隆到Ds因子的旁邻序列,其DNA顺序与亲本中Ds因子原插入位点的序列不同,表明Ds因子转座到了新的基因组位点。     

Heterologous transposon tagging of the DRL1 locus in Arabidopsis.

The development of heterologous transposon tagging systems has been an important objective for many laboratories. Here, we demonstrate the use of a Dissociation (Ds) derivative of the maize transposable element Activator (Ac) to tag the DRL1 locus of Arabidopsis. The drl1 mutant shows highly abnormal development with stunted roots, few root hairs, lanceolate leaves, and a highly enlarged, disorganized shoot apex that does not produce an inflorescence. The mutation was shown to be tightly linked to a transposed Ds, and somatic instability was observed in the presence of the transposase source. Some plants showing somatic reversion flowered and produced large numbers of wild-type progeny. These revertant progeny always inherited a DRL1 allele from which Ds had excised. Analysis of the changes in DNA sequence induced by the insertion and excision of the Ds element showed that they were typical of those induced by Ac and Ds in maize.     

Genomic and cDNA clones of the homeotic locus Antennapedia in Drosophila.

Homeotic genes are involved in the control of developmental pathways: dominant mutations at the Antennapedia locus of Drosophila, for example, lead to replacement of the antennae on the head of the fly by mesothoracic legs. Using a combination of chromosome walking and jumping, we have cloned a DNA region from Drosophila containing Antennapedia. Five DNA inversion rearrangements which are associated with the Antennapedia mutant phenotype were localized within a 25-kb region. Genomic DNA sequences from this area were used as hybridization probes to screen cDNA libraries prepared from Drosophila embryonic and pupal poly(A)+ RNA. A 2.2-kb cDNA sequence (903) was isolated which appears to derive from at least four non-contiguous chromosomal regions that span 100 kb. It includes the positions of the inversion breakpoints. A second cDNA of 2.9 kb (909) is composed of sequences from at least three chromosomal regions, two of which are similar or identical to sequences contained in the 903 clone but the third is derived from genomic DNA within a putative 903 intron. The unusual size and complexity of this locus are discussed.     

Molecular cloning of the a1 locus of Zea mays using the transposable elements En and Mu1

The a1 locus of Zea mays has been cloned using transposable elements as gene tags. The strategy was to make genomic libraries from maize stocks with a1 mutations induced either by En(Spm) or by Robertson's Mutator-system. These libraries were then screened with either Spm-I8 and En1, for the En-containing mutant, or with Mu1 for the Mu-induced mutation. There are many En and Mu1 hybridizing sequences present in the maize genome, however, by a process of cross-screening of the positives from the two libraries and by molecular analysis of the En-positive clones it was possible to identify clones in both libraries carrying all or part of the a1 gene.     

Sequences homologous to the human D1S1 locus present on human chromosome 3.

We have investigated the segregation, in somatic cell hybrids, of the human D1S1 locus, previously assigned to 1p36 by in situ hybridization. We have shown that the clone which defines this locus, lambda Ch4A-H3, originates from human chromosome 3, but contains a 1.7-kilobase (kb) PstI-HindIII repetitive element that is also present on chromosome 1, probably distal to PGD. The clone recognizes restriction fragment length polymorphisms within the single-copy sequence on chromosome 3 and one for the enzyme StuI in the repeated sequence on chromosome 1. These experiments thus expose a level of complexity in the D1S1 locus not revealed by earlier in situ hybridization studies.     

Cloning of the purA16 locus in Rec+ cells of Bacillus subtilis

A portion of purA16 chromosomal locus of Bacillus subtilis was cloned into Rec+ cells of this microorganism with pBD12 plasmid (carrying chloramphenicol and kanamycin resistance determinants) serving as a vector. The hybrid plasmids were stably maintained in cells grown on media supplemented with antibiotics and were lost from cells in the absence of drugs. The cloned fragment could incorporate into the chromosome some with a frequency of 10(-2) per cell per generation. A clone carrying the hybrid plasmid inserted into the chromosome was detected.     

Isolation and characterization of the Beadex locus of Drosophila melanogaster: a putative cis-acting negative regulatory element for the heldup-a gene.

We isolated recombinant lambda phage clones spanning 49 kilobases of DNA which contain the Beadex and heldup-a loci of Drosophila melanogaster. These cloned DNAs were used to analyze the structure of eight dominant mutant alleles of the Beadex locus which show increased gene activity. A region, only 700 base pairs in length, is altered in each of these mutants. Six of the mutations have DNA insertions within this segment. Most of these insertions resemble retrovirus-like transposable elements. In one case (Beadex2) the inserted sequences are homologous to the gypsy transposon family. The other two Beadex alleles were induced by hybrid dysgenesis and suffered deletions which included at least part of the 700-base-pair segment. These deletions appear to have resulted from imprecise excision or deletion of a nearby P element found in the wild-type parental strain. Analysis of one heldup-a allele (heldup-aD30r) indicates that a similar P element-mediated event is responsible for this lesion. In this mutant, deletion of sequences no more than 1,600 base pairs from the Beadex locus accompanies the loss of heldup-a function. The deleted sequences in heldup-aD30r include the entire 700-base-pair segment within which at least part of the Beadex locus resides, yet these flies have no Beadex phenotype. This indicates that a functional heldup-a gene is necessary for expression of the Beadex phenotype. Together, these results suggest that the Beadex functional domain is contained within a short segment of DNA near the heldup-a gene and support the hypothesis that the Beadex locus functions as a cis-acting negative regulatory element for the heldup-a gene.     

Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator.

The FATTY ACID ELONGATION1 (FAE1) gene of Arabidopsis is required for the synthesis of very long chain fatty acids in the seed. The product of the FAE1 gene is presumed to be a condensing enzyme that extends the chain length of fatty acids from C18 to C20 and C22. We report here the cloning of FAE1 by directed transposon tagging with the maize element Activator (Ac). An unstable fae1 mutant was isolated in a line carrying Ac linked to the FAE1 locus on chromosome 4. Cosegregation and reversion analyses established that the new mutant was tagged by Ac. A DNA fragment flanking Ac was cloned by inverse polymerase chain reaction and used to isolate FAE1 genomic clones and a cDNA clone from a library made from immature siliques. The predicted amino acid sequence of the FAE1 protein shares homology with those of other condensing enzymes (chalcone synthase, stilbene synthases, and beta-ketoacyl-acyl carrier protein synthase III), supporting the notion that FAE1 is the structural gene for a synthase or condensing enzyme. FAE1 is expressed in developing seed, but not in leaves, as expected from the effect of the fae1 mutation on the fatty acid compositions of those tissues.     

Isolation and characterisation of a variant allele of the gene for human apolipoprotein E

Size-selected human DNA fragments enriched in the Apolipoprotein E (ApoE) gene sequence were cloned from an individual of known ApoE phenotype, E2/E2. The clone bank was screened using a human cDNA clone for the ApoE locus (1), and a single genomic clone was isolated. Sequence data obtained from appropriate subcloned fragments confirmed that the codon for Arg-158 (CGC) in the E3 allele is altered to the codon for Cys (TGC) in the E2 allele. Hybridisation data indicated the presence of at least one intron in the ApoE gene, consistent with the structure of an independently isolated human ApoE4 allele (2).     

The Vi antigen of Salmonella typhi: molecular analysis of the viaB locus.

Strains of Salmonella typhi isolated from the blood of patients with typhoid fever invariably express a capsular polysaccharide, termed the Vi antigen. Vi antigen expression is controlled by two separate chromosomal loci, viaA and viaB. The viaA locus is commonly found in enteric bacteria. In contrast, the viaB locus appears to be specific to Vi-expressing strains of Salmonella and Citrobacter. Here the cloning, expression and analysis of viaB determinants from S. typhi Ty2 is described. Whole-cell DNA from strain Ty2 was size-fractionated and cloned into the pLA2917 cosmid vector. A recombinant cosmid, pVT1, conferring a Vi-positive phenotype upon Escherichia coli and upon the Vi-non-expressing strain Ty21a of S. typhi, was characterized and used for further studies. Transposon Tn5 insertion mutagenesis demonstrated that the Vi-antigen-encoding region on pVT1 consisted of a 15 kb fragment. A subclone, designated pVT3, which contained an 18 kb insert, was sufficient to confer Vi antigen expression upon E. coli and S. typhi Ty21a. Results of recombination experiments indicated that this DNA sequence was the viaB locus of S. typhi Ty2. In E. coli SE5000 maxicells, the viaB determinants encoded at least eight polypeptides, with molecular masses of 80, 65, 59, 48, 44, 39, 35 and 28 kDa. Functional characterization of viaB mutations in S. typhi Ty2 suggested that the 80 and 65 kDa proteins were required for cell-surface localization of the Vi antigen.