Radiological mapping of functional transcription units of bacteriophages phiX174 and S13. The function of proximal and distal promoters.

We have found that the nearest promoter is not always the primary promoter for making translatable message. The technique of ultraviolet mapping was used to determine the location of promoter sites for translated mRNA coded for by bacteriophages φX174 and S13. The method is based on the theory that the “target size” for u.v. inactivation of expression of a gene is proportional to the distance between the promoter and the 3′ end of the gene. This method has revealed an expected and some unexpected locations for the promoters responsible for gene expression. Ultraviolet-survival curves for expression of phage genes were interpreted in the following way. The contiguous genes D, F, G and H are expressed as a unit under the control of a promoter located near gene D. However, gene B (and probably the adjacent genes K and C) are controlled by a promoter distant from gene B, possibly in the region of gene H, rather than from a promoter located just before gene B. Likewise, gene A is controlled by a promoter distant from gene A.     

The size and genetic composition of virus-specific RNAs in the cytoplasm of cells producing avian sarcoma-leukosis viruses.

The genome of avian sarcoma virus (ASV) contains four known genes: gag, encoding structural proteins of the viral core; pol, encoding the viral RNA-directed DNA polymerase; env, encoding the glycoprotein(s) of the viral envelope; and src, which is responsible for neoplastic transformation of the host cell. We have located these genes on virus-specific RNAs in cells productively infected with both nondefective and defective strains of ASV by using molecular hybridization with DNAs complementary to specific portions of the ASV genome.The cytoplasm of cells producing nondefective ASV contains three species of polyadenylated virus-specific RNA, each of which has chemical polarity identical to that of the viral genome. The largest species has a molecular weight of 3.3 × 10⁶ daltons and a sedimentation coefficient of 38S, encodes all four viral genes, and is probably identical to the viral genome. A second species has a molecular weight of 1.8 × 10⁶ daltons and a sedimentation coefficient of 28S, and encodes the 3′ half of the viral genome, including env, src and a genetically silent region known as “c.” The smallest species has a molecular weight of 1.2 × 10⁶ daltons and a sedimentation coefficient of 21S, and encodes only src and “c.” All three species of virus-specific RNA contain nucleotide sequences at least partially homologous to a sequence of 101 nucleotides found at the extreme 5′ end of the ASV genome. This sequence may not be present in the portions of the ASV genome which encode the 28S and 21S virus-specific RNAs, and hence may be joined to these RNAs during their maturation from precursor molecules.The size and genetic composition of virus-specific RNAs in cells producing defective deletion mutants reflect the nature of the deletion. Deletions of either src or env eliminate the 28S virus-specific RNA, leaving a 21S RNA (which contains either env and “c” in the case of src deletions or src and “c” in the case of env deletions) and a 35S RNA which is probably identical to the viral genome.Based on these and related results, we propose a model for viral gene expression which conforms to previous suggestions that eucaryotic cells initiate translations only at the 5′ termini of messenger RNAs.     

Regulation of motility by the ExpR/Sin quorum-sensing system in Sinorhizobium meliloti

A successful symbiotic relationship between Sinorhizobium meliloti and its host Medicago sativa (alfalfa) depends on several signaling mechanisms, such as the biosynthesis of exopolysaccharides (EPS) by S. meliloti. Previous work in our laboratory has shown that a quorum-sensing mechanism controls the production of the symbiotically active EPS II. Recent microarray analysis of the whole-genome expression profile of S. meliloti reveals that the ExpR/Sin quorum-sensing system regulates additional physiological processes that include low-molecular-weight succinoglycan production, nitrogen utilization, metal transport, motility, and chemotaxis. Nearly half of the flagellar genes and their dependence on quorum sensing are prominently displayed in our microarray analyses. We extend those observations in this work and confirm the findings by real-time PCR expression analysis of selected genes, including the flaF, flbT, flaC, cheY1, and flgB genes, involved in motility and chemotaxis. These genes code for regulators of flagellum synthesis, the chemotactic response, or parts of the flagellar apparatus. Gene expression analyses and visualization of flagella by electron microscopy performed at different points in the growth phase support our proposed model in which quorum sensing downregulates motility in S. meliloti. We demonstrate that the ExpR/Sin quorum-sensing system controls motility gene expression through the VisN/VisR/Rem relay. We also show that the ExoS-dependent two-component system suppresses motility gene expression through VisN and Rem in parallel to quorum sensing. This study contributes to our understanding of the mechanisms that govern motility in S. meliloti.     

A naturally occurring insertion in the <Emphasis Type="Italic">RsFLC2</Emphasis> gene associated with late-bolting trait in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

Premature flowering reduces the yield and quality of the harvested fleshy taproot in radish. However, there has been little molecular marker research on the radish late-bolting trait. In this study, F₂ and F_2:3 populations derived from a cross of “Ninengo” (late-bolting) and “Maer” (early-bolting) were analyzed to map late-bolting genes. Five hundred insertion and deletion (InDel) markers were designed according to the whole-genome resequencing data of the two parents. A genetic map was constructed based on the F₂ population, and a late-bolting gene was detected in a 1.1-cM region between the markers InDel520 and InDel535 on chromosome R02 that explained the highest (76.4%) phenotypic variance. RsFLC2 was identified as a candidate gene in this region. Notably, “Ninengo” contains a 1627-bp insertion near the 5′ end of the first intron of RsFLC2. Allelic variation analyses in the F₂ population further validated that RsFLC2was associated with the late-bolting trait in radish. The expression pattern of RsFLC2 was significantly different between “Ninengo” and “Maer” during vernalization. Vernalization suppressed RsFLC2 expression, and the 1627-bp insertion in the first intron weakened gene repression in “Ninengo” plants, resulting in late-bolting. This study lays a foundation for uncovering the molecular mechanism of late-bolting and marker-assisted selection for breeding late-bolting varieties of radish.     

A role in true-late gene expression for the T4 bacteriophage 5' polynucleotide kinase 3' phosphatase.

Two bacteriophage T4-induced, nucleic acid-modifying activities, 5′ polynucleotide kinase and 3′ phosphatase, are both coded by the pseT gene. Therefore, the product of this gene is an enzyme which can remove phosphates from 3′ termini and add them to 5′-hydroxyl termini and thus could be said to “shuttle” phosphates on polynucleotides. This enzyme is sometimes required for T4 true-late gene expression, probably by helping establish the required intracellular DNA structure. Our data suggest that a host gene product normally can substitute for the product of the pseT gene, making it non-essential for phage multiplication on most laboratory strains of Escherichia coli.     

Analysis of expressed sequence tags from a NaHCO3-treated alkali-tolerant plant,Chloris virgata

Chloris virgata Swartz (C. virgata) is a gramineous wild plant that can survive in saline-alkali areas in northeast China. To examine the tolerance mechanisms of C. virgata, we constructed a cDNA library from whole plants of C. virgata that had been treated with 100 mM NaHCO₃ for 24 h and sequenced 3168 randomly selected clones. Most (2590) of the expressed sequence tags (ESTs) showed significant similarity to sequences in the NCBI database. Of the 2590 genes, 1893 were unique. Gene Ontology (GO) Slim annotations were obtained for 1081 ESTs by BLAST2GO and it was found that 75 genes of them were annotated with GO terms “response to stress”, “response to abiotic stimulus”, and “response to biotic stimulus”, indicating these genes were likely to function in tolerance mechanism of C. virgata.In a separate experiment, 24 genes that are known from previous studies to be associated with abiotic stress tolerance were further examined by real-time RT-PCR to see how their expressions were affected by NaHCO₃ stress. NaHCO₃ treatment up-regulated the expressions of pathogenesis-related gene (DC998527), Win1 precursor gene (DC998617), catalase gene (DC999385), ribosome inactivating protein 1 (DC999555), Na⁺/H⁺ antiporter gene (DC998043), and two-component regulator gene (DC998236).     

Tightly Regulated Expression of Autographa californica Multicapsid Nucleopolyhedrovirus Immediate Early Genes Emerges from Their Interactions and Possible Collective Behaviors

To infect their hosts, DNA viruses must successfully initiate the expression of viral genes that control subsequent viral gene expression and manipulate the host environment. Viral genes that are immediately expressed upon infection play critical roles in the early infection process. In this study, we investigated the expression and regulation of five canonical regulatory immediate-early (IE) genes of Autographa californica multicapsid nucleopolyhedrovirus: ie0, ie1, ie2, me53, and pe38. A systematic transient gene-expression analysis revealed that these IE genes are generally transactivators, suggesting the existence of a highly interactive regulatory network. A genetic analysis using gene knockout viruses demonstrated that the expression of these IE genes was tolerant to the single deletions of activator IE genes in the early stage of infection. A network graph analysis on the regulatory relationships observed in the transient expression analysis suggested that the robustness of IE gene expression is due to the organization of the IE gene regulatory network and how each IE gene is activated. However, some regulatory relationships detected by the genetic analysis were contradictory to those observed in the transient expression analysis, especially for IE0-mediated regulation. Statistical modeling, combined with genetic analysis using knockout alleles for ie0 and ie1, showed that the repressor function of ie0 was due to the interaction between ie0 and ie1, not ie0 itself. Taken together, these systematic approaches provided insight into the topology and nature of the IE gene regulatory network.     

Control of MarRAB Operon in Escherichia coli via Autoactivation and?Autorepression

Choice of network topology for gene regulation has been a question of interest for a long time. How do simple and more complex topologies arise? In this work, we analyze the topology of the marRAB operon in Escherichia coli, which is associated with control of expression of genes associated with conferring resistance to low-level antibiotics to the bacterium. Among the 2102 promoters in E. coli, the marRAB promoter is the only one that encodes for an autoactivator and an autorepressor. What advantages does this topology confer to the bacterium? In this work, we demonstrate that, compared to control by a single regulator, the marRAB regulatory arrangement has the least control cost associated with modulating gene expression in response to environmental stimuli. In addition, the presence of dual regulators allows the regulon to exhibit a diverse range of dynamics, a feature that is not observed in genes controlled by a single regulator.     

Similar tissue-specific expression of the Adh genes from different Drosophila species is mediated by distinct arrangements of cis-acting sequences

In Drosophila melanogaster transformants, the alcohol dehydrogenase (Adh) genes from D. affinidisjuncta and D. grimshawi show similar levels of expression except in the adult midgut where the D. affinidisjuncta gene is expressed about 10- to 20-fold more strongly. To study the arrangement of cis-acting sequences responsible for this regulatory difference, homologous restriction sites were used to create a series of chimeric genes that switched fragments from the 5′ and 3′ flanking regions of these two genes. Chimeric genes were introduced into the germ-line of D. melanogaster, and Adh gene expression was analyzed by measuring RNA levels. Various gene fragments in the promoter region and elsewhere influence expression in the adult midgut and in whole larvae and adults. Comparison of these results with earlier studies involving chimeras between the D. affinidisjuncta and D. hawaiiensis genes indicates that expression in the adult midgut is influenced by multiple regulatory sequences and that distinct arrangements of regulatory sequences can result in similar levels of expression both in the adult midgut and in the whole organism.