Modulation of lipid metabolism and spiramycin biosynthesis in Streptomyces ambofaciens unstable mutants.

Streptomyces ambofaciens is prone to genetic instability involving genomic rearrangements at the extremities of the chromosomal DNA. An amplified DNA sequence (ADS205), including an open reading frame (orfPS), is responsible for the reversible loss of spiramycin production in the mutant strain NSA205 (ADS205(+) Spi-). The product of orfPS is homologous to polyketide synthase systems (PKSs) involved in the biosynthesis of erythromycin and rapamycin and is overexpressed in strain NSA205 compared with the parental strain RP181110. As PKSs and fatty acid synthase systems have the same precursors, we tested the possibility that overexpression of orfPS also affects lipid metabolism in strain NSA205. This report focuses on comparative analysis of lipids in strain RP181110, the mutant strain NSA205, and a derivative, NSA228 (ADS205(-) Spi+). NSA205 showed a dramatically depressed lipid content consisting predominantly of phospholipids and triacylglycerols. This lipid content was globally restored in strain NSA228, which had lost ADS205. Furthermore, strains RP181110 and NSA205 presented similar phospholipid and triacylglycerol compositions. No abnormal fatty acids were detected in NSA205.     

Genetic instability and associated genome plasticity in Streptomyces ambofaciens: pulsed-field gel electrophoresis evidence for large DNA alterations in a limited genomic region.

Using pulsed-field gel electrophoresis (PFGE) analysis, the amplifiable units of DNA (AUD) loci AUD6 and AUD90 of Streptomyces ambofaciens DSM40697 could be mapped in the wild-type genome within two adjacent AseI restriction fragments estimated to be about 75 and 850 kb. In addition, the genetic instability and formation of very large deletions were strictly correlated. Their sizes were estimated to range from 250 to more than 2,000 kb. These deletions affected the DNA region overlapping both amplifiable loci. PFGE also allowed us to localize the amplified DNA sequences and to establish their structure: amplification takes place at the AUD locus as a tandem array of the wild-type AUD sequence.     

DNA rearrangements at the extremities of the Streptomyces ambofaciens linear chromosome: evidence for developmental control

In Streptomyces, a genomic instability results from frequent recombination events which occur at the ends of the linear chromosomal DNA. These events are believed to be responsible for the variability observed in these regions among Streptomyces species and strains. In order to identify functions able to control this type of genome plasticity, mutators as well as mutants produced at different stages of development have been characterized in S. ambofaciens. Their characterization suggests the existence of a relationship between genomic instability and colony development.     

The unstable region of Streptomyces ambofaciens includes 210 kb terminal inverted repeats flanking the extremities of the linear chromosomal DNA

Physical maps of the chromosomes of three strains of Streptomyces ambofaciens were constructed by ordering Ase I fragments generated from the genomic DNA as a single linear chromosome of about 8 Mb. The physical maps of the three strains were very similar. For strain DSM40697, a Dra I map was obtained by positioning the Dra I sites relative to the Ase I map. Eighteen genetic markers as well as the deletable and amplifiable region were assigned to the Ase I and Dra I fragments in this strain. The resulting genetic map resembled that of Streptomyces coelicolor A3(2). The twoterminal Ase I fragments exhibited retarded pulsed-field gel electrophoresis mobility, demonstrating that proteins are covalently bound at this position. A restriction map of this region was made using four additional endonucleases. Repeated sequences present at both ends of the chromosome were mapped as long terminal inverted repeats stretching over 210 kb. This corresponds to the longest terminal inverted repeats so far characterized. The deletable region of S. ambofaciens was localized at the chromosomal extremities.     

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.     

Cloning and characterization of a regulatory gene of the SARP family and its flanking region from Streptomyces ambofaciens

In a search for activators of secondary metabolism we isolated a 12.6-kb DNA fragment from a genomic library of Streptomyces ambofaciens NRRL 2240 (the spiramycin producer ). Sequencing of 6 kb of the cloned fragment revealed a cluster of four ORFs (ORF1–4) whose deduced products showed similarities to those of other genes involved in polyketide biosynthesis, including a pathway-specific regulatory gene of the SARP family. The results of insertional inactivation of some of the cloned genes clearly indicate that the isolated cluster does not code for spiramycin production, suggesting that some other polyketide compound might well be produced by this strain. Received: 14 May 1999 / Accepted: 28 July 1999     

Changes in reproductive roles are associated with changes in gene expression in fire ant queens

In species with social hierarchies, the death of dominant individuals typically upheaves the social hierarchy and provides an opportunity for subordinate individuals to become reproductives. Such a phenomenon occurs in the monogyne form of the fire ant, Solenopsis invicta, where colonies typically contain a single wingless reproductive queen, thousands of workers and hundreds of winged nonreproductive virgin queens. Upon the death of the mother queen, many virgin queens shed their wings and initiate reproductive development instead of departing on a mating flight. Workers progressively execute almost all of them over the following weeks. To identify the molecular changes that occur in virgin queens as they perceive the loss of their mother queen and begin to compete for reproductive dominance, we collected virgin queens before the loss of their mother queen, 6 h after orphaning and 24 h after orphaning. Their RNA was extracted and hybridized against microarrays to examine the expression levels of approximately 10 000 genes. We identified 297 genes that were consistently differentially expressed after orphaning. These include genes that are putatively involved in the signalling and onset of reproductive development, as well as genes underlying major physiological changes in the young queens.     

Integrated genomic and gene expression profiling identifies two major genomic circuits in urothelial carcinoma

Similar to other malignancies, urothelial carcinoma (UC) is characterized by specific recurrent chromosomal aberrations and gene mutations. However, the interconnection between specific genomic alterations, and how patterns of chromosomal alterations adhere to different molecular subgroups of UC, is less clear. We applied tiling resolution array CGH to 146 cases of UC and identified a number of regions harboring recurrent focal genomic amplifications and deletions. Several potential oncogenes were included in the amplified regions, including known oncogenes like E2F3, CCND1, and CCNE1, as well as new candidate genes, such as SETDB1 (1q21), and BCL2L1 (20q11). We next combined genome profiling with global gene expression, gene mutation, and protein expression data and identified two major genomic circuits operating in urothelial carcinoma. The first circuit was characterized by FGFR3 alterations, overexpression of CCND1, and 9q and CDKN2A deletions. The second circuit was defined by E3F3 amplifications and RB1 deletions, as well as gains of 5p, deletions at PTEN and 2q36, 16q, 20q, and elevated CDKN2A levels. TP53/MDM2 alterations were common for advanced tumors within the two circuits. Our data also suggest a possible RAS/RAF circuit. The tumors with worst prognosis showed a gene expression profile that indicated a keratinized phenotype. Taken together, our integrative approach revealed at least two separate networks of genomic alterations linked to the molecular diversity seen in UC, and that these circuits may reflect distinct pathways of tumor development.     

Recurrent reciprocal genomic rearrangements of 17q12 are associated with renal disease, diabetes, and epilepsy

Most studies of genomic disorders have focused on patients with cognitive disability and/or peripheral nervous system defects. In an effort to broaden the phenotypic spectrum of this disease model, we assessed 155 autopsy samples from fetuses with well-defined developmental pathologies in regions predisposed to recurrent rearrangement, by array-based comparative genomic hybridization. We found that 6% of fetal material showed evidence of microdeletion or microduplication, including three independent events that likely resulted from unequal crossing-over between segmental duplications. One of the microdeletions, identified in a fetus with multicystic dysplastic kidneys, encompasses the TCF2 gene on 17q12, previously shown to be mutated in maturity-onset diabetes, as well as in a subset of pediatric renal abnormalities. Fine-scale mapping of the breakpoints in different patient cohorts revealed a recurrent 1.5-Mb de novo deletion in individuals with phenotypes that ranged from congenital renal abnormalities to maturity-onset diabetes of the young type 5. We also identified the reciprocal duplication, which appears to be enriched in samples from patients with epilepsy. We describe the first example of a recurrent genomic disorder associated with diabetes.     

Strain-related differences in antibody-mediated changes in gene expression are associated with differences in capsule and location of binding

We recently established that antibody (Ab)-binding can induce gene expression changes in a serotype A strain (H99) of the pathogenic yeast, Cryptococcus neoformans. That study showed that monoclonal antibodies (mAbs) differing in epitope specificity and protective efficacy elicited differences in gene expression. Because many mAbs bind to serotypes A and D strains differently, we now investigate the binding of one mAb to two strains representing these serotypes. Cells of the serotype A strain H99 and the serotype D strain 24067 were incubated with near saturating concentrations of the IgG1 capsule-binding mAb 18B7 or MOPC, an irrelevant mAb matched control. Comparative immunofluorescence analysis of mAb 18B7 binding revealed that it bound closer to the cell wall in H99 than 24067, where it was associated with decreased or increased cell diameter, respectively. A comparison of encapsulated cell compressibility showed that strain 24067 was more compressible than that of strain H99. RNA was extracted and used for gene expression analysis using the C. neoformans JEC21 genomic microarray. After 1h incubation with mAb 18B7, there were just 2 gene expression changes observed with strain 24067 or strain JEC21, unlike the 43 seen with strain H99. After 4h incubation with mAb 18B7, there were 14 and 140 gene expression changes observed with strain 24067 and JEC21, respectively. Thus, C. neoformans strains differ both in the response and the time of response to mAb binding and these differences may reflect differences in the location of Ab binding, Ab-mediated changes in cell diameter and compressibility of the capsular polysaccharide.     

Primary structure analysis of a duplicated region in the amplifiable AUD6 locus of Streptomyces ambofaciens DSM40697

Abstract In Streptomyces ambofaciens , an amplifiable unit of DNA ( AUD6 ) contains two homologous sequences, one located on the right extremity of the AUD ( S1R ), the other being internal ( IHS ). This paper presents the molecular analysis of this duplication. The nucleotide sequences are almost identical (95%) and each contains an ORF of about 330 codons, the two ORFs being nearly identical. The two hypothetical proteins, deduced from these sequences, show about 30% identity with different bacterial repressors. They also show a particularly strong similarity (90% identity between the full-length sequences) with hypothetical proteins of Streptomyces lividans 66 encoded by sequences also present on an amplifiable DNA region ( AUD1 ).     

Coordinate changes in Myosin heavy chain isoform gene expression are selectively associated with alterations in dilated cardiomyopathy phenotype

BACKGROUND: The most common cause of chronic heart failure in the US is secondary or primary dilated cardiomyopathy (DCM). The DCM phenotype exhibits changes in the expression of genes that regulate contractile function and pathologic hypertrophy. However, it is unclear if any of these alterations in gene expression are disease producing or modifying. MATERIALS AND METHODS: One approach to providing evidence for cause-effect of a disease-influencing gene is to quantitatively compare changes in phenotype to changes in gene expression by employing serial measurements in a longitudinal experimental design. We investigated the quantitative relationships between changes in gene expression and phenotype n 47 patients with idiopathic DCM. In endomyocardial biopsies at baseline and 6 months later, we measured mRNA expression of genes regulating contractile function (beta-adrenergic receptors, sarcoplasmic reticulum Ca(2) + ATPase, and alpha- and beta-myosin heavy chain isoforms) or associated with pathologic hypertrophy (beta-myosin heavy chain and atrial natriuretic peptide), plus beta-adrenergic receptor protein expression. Left ventricular phenotype was assessed by radionuclide ejection fraction. RESULTS: Improvement in DCM phenotype was directly related to a coordinate increase in alpha- and a decrease in beta-myosin heavy chain mRNA expression. In contrast, modification of phenotype was unrelated to changes in the expression of beta(1)- or beta(2)-adrenergic receptor mRNA or protein, or to the mRNA expression of sarcoplasmic reticulum Ca(2) + ATPase and atrial natriuretic peptide. CONCLUSION: We conclude that in human DCM, phenotypic modification is selectively associated with myosin heavy chain isoform changes. These data support the hypothesis that myosin heavy chain isoform changes contribute to disease progression in human DCM.     

Large scale comparison of global gene expression patterns in human and mouse

Background  

It is widely accepted that orthologous genes between species are conserved at the sequence level and perform similar functions in different organisms. However, the level of conservation of gene expression patterns of the orthologous genes in different species has been unclear. To address the issue, we compared gene expression of orthologous genes based on 2,557 human and 1,267 mouse samples with high quality gene expression data, selected from experiments stored in the public microarray repository ArrayExpress.