Unstable inheritance of maize B-type chromosomes that lack centric heterochromatin

The B chromosome of maize undergoes frequent non-disjunction at the second pollen mitosis. In B–A translocations, the B–A chromosome retains the capacity for non-disjunction. We have collected deletion-derivative TB-9Sb stocks. One derivative, the "type 1 telocentric", has a B–9 chromosome that lacks centric heterochromatin. It produces few recessive (non-disjunctional) phenotypes in pollen parent testcrosses of the translocation heterozygote, 9 9–B telo B–9. The finding helped demonstrate the role of centric heterochromatin in non-disjunction. An isochromo some derivative of the type 1 telocentric was also recovered. It was tested in the 9–B 9–B iso B–9 constitution. This is equivalent to 9 9–B telo B–9 in terms of chromosome 9 dosage. Surprisingly, crosses with the isochromosome gave significant levels of recessive phenotypes. In addition, high levels of variegated phenotypes were found. Recently, a circumstance was found that makes inheritance of the type 1 telocentric chromosome somewhat similar to that of the isochromosome. Crosses with hypoploid 9–B 9–B telo B–9 plants showed significant levels of recessive and variegated phenotypes. These crosses were investigated to help explain the source(s) of the phenotypes. Cytological and genetic studies were performed. Centric misdivision was found to account for the variegated phenotypes. A mixture of conventional B non-disjunction and centric misdivision produced the recessive phenotypes. The significance of conventional non-disjunction in the absence of centric heterochromatin is discussed.Key words: cytogenetics, B chromosome, centromere, maize.

Four Loci on Abnormal Chromosome 10 Contribute to Meiotic Drive in Maize

We provide a genetic analysis of the meiotic drive system on maize abnormal chromosome 10 (Ab10) that causes preferential segregation of specific chromosomal regions to the reproductive megaspore. The data indicate that at least four chromosomal regions contribute to meiotic drive, each providing distinct functions that can be differentiated from each other genetically and/or phenotypically. Previous reports established that meiotic drive requires neocentromere activity at specific tandem repeat arrays (knobs) and that two regions on Ab10 are involved in trans-activating neocentromeres. Here we confirm and extend data suggesting that only one of the neocentromere-activating regions is sufficient to move many knobs. We also confirm the localization of a locus/loci on Ab10, thought to be a prerequisite for meiotic drive, which promotes recombination in structural heterozygotes. In addition, we identified two new and independent functions required for meiotic drive. One was identified through the characterization of a deletion derivative of Ab10 [Df(L)] and another as a newly identified meiotic drive mutation (suppressor of meiotic drive 3). In the absence of either function, meiotic drive is abolished but neocentromere activity and the recombination effect typical of Ab10 are unaffected. These results demonstrate that neocentromere activity and increased recombination are not the only events required for meiotic drive.

FpvA Receptor Involvement in Pyoverdine Biosynthesis in Pseudomonas aeruginosa

ABSTRACT Alignment of the Pseudomonas aeruginosa ferric pyoverdine receptor, FpvA, with similar ferric-siderophore receptors revealed that the mature protein carries an extension of ca. 70 amino acids at its N terminus, an extension shared by the ferric pseudobactin receptors of P. putida. Deletion of fpvA from the chromosome of P. aeruginosa reduced pyoverdine production in this organism, as a result of a decline in expression of genes (e.g., pvdD) associated with the biosynthesis of the pyoverdine peptide moiety. Wild-type fpvA restored pvd expression in the mutant, thereby complementing its pyoverdine deficiency, although a deletion derivative of fpvA encoding a receptor lacking the N terminus of the mature protein did not. The truncated receptor was, however, functional in pyoverdine-mediated iron uptake, as evidenced by its ability to promote pyoverdine-dependent growth in an iron-restricted medium. These data are consistent with the idea that the N-terminal extension plays a role in FpvA-mediated pyoverdine biosynthesis in P. aeruginosa.

Complementation of Conjugation Functions of Streptomyces lividans Plasmid pIJ101 by the Related StreptomycesPlasmid pSB24.2

ABSTRACT A database search revealed extensive sequence similarity betweenStreptomyces lividans plasmid pIJ101 andStreptomyces plasmid pSB24.2, which is a deletion derivative of Streptomyces cyanogenus plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, thecis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of complementing the pIJ101 clt and korBfunctions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems.

Potential of Conjugal Transfer as a Strategy for the Introduction of Recombinant Genetic Material into Strains ofLactobacillus helveticus

ABSTRACT Cointegrates generated between a plasmid pIP501 deletion derivative (pVA797) and nonconjugative shuttle vector pSA3 were confirmed as capable of exconjugation from lactococci into a range of strains ofLactobacillus helveticus with the concomitant expression of a recombinant gene. The plasmid cointegrate that was formed appeared to be segregationally stable at 37°C in some host strains. In all strains, however, the plasmid became increasingly unstable as the incubation temperature was raised. The technique offers not only a generalized method for the introduction of novel genetic material into this important industrial microbe but also the possibility of exploiting the thermal sensitivity of the plasmid to enable it to act as a delivery system for the integration of cloned genes into the bacterial chromosome, at restrictive temperatures, by recombination at regions of homology.

Functional Analysis of Deletion Derivatives of the Maize Transposon MuDR Delineates Roles for the MURA and MURB Proteins

The regulatory transposon of the Mutator system of transposable elements in maize is MuDR. MuDR elements produce two transcripts, from genes mudrA and mudrB, encoding proteins MURA and MURB, respectively. Like many other transposons, MuDR elements often undergo deletions, usually of internal sequences. Analysis of a deletion that is restricted to the region encoding MURB demonstrates that this gene is not required to cause excisions of a reporter element, although it may be required for transposition or suppression of suppressible alleles. Conversely, a derivative that lacks the region encoding MURA but that produces MURB is nonfunctional for all aspects of Mutator activity. Northern analysis of these derivatives reveals that each of the two transcripts can be independently transcribed, and analysis using an antibody specific for MURB reveals that mudrB transcript can also be successfully translated and its product appropriately localized in the absence of mudrA. A third deletion derivative provides evidence for a source of previously reported antisense transcript.

Transcriptional Regulation of theStreptococcus salivarius 57.I Urease Operon

ABSTRACT The Streptococcus salivarius 57.I urecluster was organized as an operon, beginning withureI, followed by ureABC (structural genes) andureEFGD (accessory genes). Northern analyses revealed transcripts encompassing structural genes and transcripts containing the entire operon. A ς70-like promoter could be mapped 5′ to ureI (PureI) by primer extension analysis. The intensity of the signal increased when cells were grown at an acidic pH and was further enhanced by excess carbohydrate. To determine the function(s) of two inverted repeats located 5′ toPureI, transcriptional fusions of the full-length promoter region (PureI), or a deletion derivative (PureIΔ100), and a promoterless chloramphenicol acetyltransferase (CAT) gene were constructed and integrated into the chromosome to generate strains PureICAT andPureIΔ100CAT, respectively. CAT specific activities ofPureICAT were repressed at pH 7.0 and induced at pH 5.5 and by excess carbohydrate. In PureIΔ100CAT, CAT activity was 60-fold higher than in PureICAT at pH 7.0 and pH induction was nearly eliminated, indicating that expression was negatively regulated. Thus, it was concluded that PureI was the predominant, regulated promoter and that regulation was governed by a mechanism differing markedly from other known mechanisms for bacterial urease expression.

Construction of a Stable Attenuated Shigella sonnei ΔvirG Vaccine Strain, WRSS1, and Protective Efficacy and Immunogenicity in the Guinea Pig Keratoconjunctivitis Model

ABSTRACT Construction of a stable Shigella sonnei vaccine has been complicated by the instability of the virulence phenotype caused by the spontaneous loss of the invasion plasmid. To select a suitable candidate for vaccine construction, 16 S. sonneistrains were screened for stability of the virulence phenotype. A stable strain, S. sonnei Mosely, was selected for further work. pΔvirG2, a deletion derivative of the virGgene in the sacB suicide vector pCVD442, was used to generate an S. sonnei virG deletion strain, WRSS1, which was invasive in HeLa cells but negative in the Sereny test. WRSS1 was found to be both immunogenic and protective in the guinea pig keratoconjunctivitis model.

Immunohistological Localization of the MrkD Adhesin in the Type 3 Fimbriae of Klebsiella pneumoniae

ABSTRACT The adhesive minor protein MrkD of the type 3 fimbria ofKlebsiella pneumoniae was expressed and purified fromEscherichia coli as a fusion protein with an N-terminal polyhistidine tail. Polyclonal antibodies raised against MrkD specifically recognized the MrkD peptide in Western blots of fimbrial preparations. Immunoelectron microscopic analyses showed that the anti-MrkD immunoglobulins bound to the tip of the plasmid-encoded variant of the type 3 fimbria of K. pneumoniae, whereas no binding to the chromosomally encoded MrkD-deficient type 3 fimbrial variant of K. pneumoniae was detected. Immunoglobulins from an antiserum raised against purified type 3 fimbrial filaments bound laterally to both type 3 fimbrial variants. The anti-MrkD antibodies also bound to the tip of a papG deletion derivative of theE. coli P fimbria complemented with mrkD, indicating that MrkD structurally complements a PapG mutation in the P fimbria of E. coli.

Epigenetic Control of a Transposon-Inactivated Gene in Neurospora is Dependent on DNA Methylation

An unstable allele of the Neurospora am (GDH) gene resulting from integration of the retrotransposon Tad3-2 into 5′ noncoding sequences was found in previous work. We report that reversion to Am+ depends on DNA methylation within and upstream of Tad. Levels of methylation were correlated with the proportion of Am+ conidia, whether the cultures were derived from Am− or Am+ isolates. Reversion to Am+ did not occur when conidia were plated on 5-azacytidine, which reduces DNA methylation. The mutation dim-2, which appears to abolish DNA methylation, also prevented reversion to Am+. The native am allele, in a strain that lacked Tad elements, was replaced with am:: Tad3-2 or with a deletion derivative that prevents transposition of Tad. Transformants of both classes showed instability comparable with that of the original isolates, which contain multiple Tad elements. Deletion of the upstream enhancer-like sequences, URSamα and β, did not prevent the instability of am:: Tad3-2. The results suggest that am expression is dependent on DNA methylation but not on proliferation or transposition of the Tad element and that the instability does not require the upstream sequences of am.