Identification of Haemophilus influenzae Rd transformation genes using cassette mutagenesis.
(25/7552)
Genes required for natural transformation of Haemophilus influenzae Rd were identified by a cassette mutagenesis protocol consisting of the following steps: random insertional mutagenesis, phenotypic screening, sequencing of genome sequence tags from the DNA flanking the insertion in the selected mutants and comparison of genome sequence tags to genomic sequence data. The cassette mutagenesis screen for transformation genes resulted in five distinct mutant classes, two of which have been identified in previous studies. Insertions in the three newly identified loci interrupted genes with predicted protein products homologous to a type IV pilin-like protein biogenesis operon, drug-efflux transporters and a phospholipid-biosynthesis enzyme. The most significant finding of this screen is the requirement for type IV pilin-like proteins in genetic transformation of H. influenzae. These surface structures are utilized for DNA uptake in a number of Gram-positive and Gram-negative bacteria, and appear to be the common component among the systems for DNA binding. (+info)
Position-dependent inhibition of class-switch recombination by PGK-neor cassettes inserted into the immunoglobulin heavy chain constant region locus.
(26/7552)
The Ig heavy chain (IgH) constant region (CH) genes are organized from 5' to 3' in the order Cmicro, Cdelta, Cgamma3, Cgamma1, Cgamma2b, Cgamma2a, Cepsilon, and Calpha. Expression of CH genes downstream of Cdelta involves class-switch recombination (CSR), a process that is targeted by germ-line transcription (GT) of the corresponding CH gene. Previously, we demonstrated that insertion of a PGK-neor cassette at two sites downstream of Calpha inhibits, in cultured B cells, GT of and CSR to a subset of CH genes (including Cgamma3, Cgamma2a, Cgamma2b, and Cepsilon) that lie as far as 120 kb upstream. Here we show that insertion of the PGK-neor cassette in place of sequences in the Igamma2b locus inhibits GT of and CSR to the upstream Cgamma3 gene, but has no major effect on the downstream Cgamma2a and Cepsilon genes. Moreover, replacement of the Cepsilon exons with a PGK-neor cassette in the opposite transcriptional orientation also inhibits, in culture, GT of and CSR to the upstream Cgamma3, Cgamma2b, and Cgamma2a genes. As with the PGK-neor insertions 3' of Calpha studied previously, the Cgamma1 and Calpha genes were less affected by these mutations both in culture and in mice, whereas the Cgamma2b gene appeared less affected in vivo. Our findings support the existence of a long-range 3' IgH regulatory region required for GT of and CSR to multiple CH genes and suggest that PGK-neor cassette insertion into the locus short circuits the ability of this region to facilitate GT of dependent CH genes upstream of the insertion. (+info)
The Drosophila beta FTZ-F1 orphan nuclear receptor provides competence for stage-specific responses to the steroid hormone ecdysone.
(27/7552)
The acquisition of competence is a key mechanism for refining global signals to distinct spatial and temporal responses. The molecular basis of competence, however, remains poorly understood. Here, we show that the beta FTZ-F1 orphan nuclear receptor functions as a competence factor for stage-specific responses to the steroid hormone ecdysone during Drosophila metamorphosis. beta FTZ-F1 mutants pupariate normally in response to the late larval pulse of ecdysone but display defects in stage-specific responses to the subsequent ecdysone pulse in prepupae. The ecdysone-triggered genetic hierarchy that directs these developmental responses is severely attenuated in beta FTZ-F1 mutants, although ecdysone receptor expression is unaffected. This study define beta FTZ-F1 as an essential competence factor for stage-specific responses to a steroid signal and implicates interplay among nuclear receptors as a mechanism for achieving hormonal competence. (+info)
Subtle mutagenesis by ends-in recombination in malaria parasites.
(28/7552)
The recent advent of gene-targeting techniques in malaria (Plasmodium) parasites provides the means for introducing subtle mutations into their genome. Here, we used the TRAP gene of Plasmodium berghei as a target to test whether an ends-in strategy, i.e., targeting plasmids of the insertion type, may be suitable for subtle mutagenesis. We analyzed the recombinant loci generated by insertion of linear plasmids containing either base-pair substitutions, insertions, or deletions in their targeting sequence. We show that plasmid integration occurs via a double-strand gap repair mechanism. Although sequence heterologies located close (less than 450 bp) to the initial double-strand break (DSB) were often lost during plasmid integration, mutations located 600 bp and farther from the DSB were frequently maintained in the recombinant loci. The short lengths of gene conversion tracts associated with plasmid integration into TRAP suggests that an ends-in strategy may be widely applicable to modify plasmodial genes and perform structure-function analyses of their important products. (+info)
The neuroendocrine protein 7B2 is required for peptide hormone processing in vivo and provides a novel mechanism for pituitary Cushing's disease.
(29/7552)
The neuroendocrine protein 7B2 has been implicated in activation of prohormone convertase 2 (PC2), an important neuroendocrine precursor processing endoprotease. To test this hypothesis, we created a null mutation in 7B2 employing a novel transposon-facilitated technique and compared the phenotypes of 7B2 and PC2 nulls. 7B2 null mice have no demonstrable PC2 activity, are deficient in processing islet hormones, and display hypoglycemia, hyperproinsulinemia, and hypoglucagonemia. In contrast to the PC2 null phenotype, these mice show markedly elevated circulating ACTH and corticosterone levels, with adrenocortical expansion. They die before 9 weeks of severe Cushing's syndrome arising from pituitary intermediate lobe ACTH hypersecretion. We conclude that 7B2 is indeed required for activation of PC2 in vivo but has additional important functions in regulating pituitary hormone secretion. (+info)
The Mycobacterium tuberculosis recA intein can be used in an ORFTRAP to select for open reading frames.
(30/7552)
The DNA repair protein RecA of Mycobacterium tuberculosis contains an intein, a self-splicing protein element. We have employed this Mtu recA intein to create a selection system for successful intein splicing by inserting it into a kanamycin-resistance gene so that functional antibiotic resistance can only be restored upon protein splicing. We then proceeded to develop an ORFTRAP, i.e., a selection system for the cloning of open reading frames (ORFs). The ORFTRAP exploits the self-splicing properties of inteins (which depend on full-length in-frame translation of a precursor protein) by allowing protein splicing to occur when DNA fragments encoding ORFs are inserted into the Mtu recA intein, whereas DNA fragments containing non-ORFs are selected against. Regions of the Mtu recA intein that tolerate the insertion of additional amino acids were identified by Bgl II linker scanning mutagenesis, and a respective construct was chosen as the ORFTRAP. To test the maximum insert size that could be cloned into ORFTRAP, DNA fragments of increasing length from the Listeria monocytogenes hly gene as well as a genomic library of Haemophilus influenzae were inserted and it was found that the longest permissive inserts were 425 bp and 251 bp, respectively. The H. influenzae ORFTRAP library also demonstrated the strength (strong selection power) and weakness (insertion of very small fragments) of the system. Further modifications should make the ORFTRAP useful for protein expression, epitope mapping, and antigen screening. (+info)
Characterization of the pyoluteorin biosynthetic gene cluster of Pseudomonas fluorescens Pf-5.
(31/7552)
Ten genes (plt) required for the biosynthesis of pyoluteorin, an antifungal compound composed of a bichlorinated pyrrole linked to a resorcinol moiety, were identified within a 24-kb genomic region of Pseudomonas fluorescens Pf-5. The deduced amino acid sequences of eight plt genes were similar to the amino acid sequences of genes with known biosynthetic functions, including type I polyketide synthases (pltB, pltC), an acyl coenzyme A (acyl-CoA) dehydrogenase (pltE), an acyl-CoA synthetase (pltF), a thioesterase (pltG), and three halogenases (pltA, pltD, and pltM). Insertions of the transposon Tn5 or Tn3-nice or a kanamycin resistance gene in each of these genes abolished pyoluteorin production by Pf-5. The presumed functions of the eight plt products are consistent with biochemical transformations involved in pyoluteorin biosynthesis from proline and acetate precursors. Isotope labeling studies demonstrated that proline is the primary precursor to the dichloropyrrole moiety of pyoluteorin. The deduced amino acid sequence of the product of another plt gene, pltR, is similar to those of members of the LysR family of transcriptional activators. pltR and pltM are transcribed divergently from the pltLABCDEFG gene cluster, and a sequence with the characteristics of a LysR binding site was identified within the 486-bp intergenic region separating pltRM from pltLABCDEFG. Transcription of the pyoluteorin biosynthesis genes pltB, pltE, and pltF, assessed with transcriptional fusions to an ice nucleation reporter gene, was significantly greater in Pf-5 than in a pltR mutant of Pf-5. Therefore, PltR is proposed to be a transcriptional activator of linked pyoluteorin biosynthesis genes. (+info)
Genetic localization and molecular characterization of two key genes (mitAB) required for biosynthesis of the antitumor antibiotic mitomycin C.
(32/7552)
Mitomycin C (MC) is an antitumor antibiotic derived biosynthetically from 3-amino-5-hydroxybenzoic acid (AHBA), D-glucosamine, and carbamoyl phosphate. A gene (mitA) involved in synthesis of AHBA has been identified and found to be linked to the MC resistance locus, mrd, in Streptomyces lavendulae. Nucleotide sequence analysis showed that mitA encodes a 388-amino-acid protein that has 71% identity (80% similarity) with the rifamycin AHBA synthase from Amycolatopsis mediterranei, as well as with two additional AHBA synthases from related ansamycin antibiotic-producing microorganisms. Gene disruption and site-directed mutagenesis of the S. lavendulae chromosomal copy of mitA completely blocked the production of MC. The function of mitA was confirmed by complementation of an S. lavendulae strain containing a K191A mutation in MitA with AHBA. A second gene (mitB) encoding a 272-amino-acid protein (related to a group of glycosyltransferases) was identified immediately downstream of mitA that upon disruption resulted in abrogation of MC synthesis. This work has localized a cluster of key genes that mediate assembly of the unique mitosane class of natural products. (+info)