The intron-containing gene for yeast profilin (PFY) encodes a vital function

The gene coding for profilin (PFY), an actin-binding protein, occurs as a single copy in the haploid genome of Saccharomyces cerevisiae and is required for spore germination and cell viability. Displacement of one gene copy in a diploid cell by a nonfunctional allele is recessively lethal: tetrad analysis yields only two viable spores per ascus. The PFY gene maps on chromosome XV and is linked to the ADE2 marker. The primary transcript of about 1,000 bases contains an intron of 209 bases and is spliced into a messenger of about 750 bases. The intron was identified by comparison with a cDNA clone, which also revealed the 3' end of the transcript. The 5' end of the mRNA was mapped by primer elongation. The gene is transcribed constitutively and has a coding capacity for a protein of 126 amino acids. The deduced molecular weight of

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Coordinate expression of amplified metallothionein I and II genes in cadmium-resistant Chinese hamster cells

Molecular and Cellular Biology ◽

10.1128/mcb.5.12.3525-3531.1985 ◽

1985 ◽

Vol 5 (12) ◽

pp. 3525-3531

Author(s):

J K Griffith

Keyword(s):

Cell Line ◽

Cell Lines ◽

Recombinant Dna ◽

Chinese Hamster ◽

Single Copy ◽

Ovary Cell ◽

Gene Copy ◽

Mrna Levels ◽

Protein Levels ◽

Copy Numbers

Recombinant DNA probes complementary to Chinese hamster metallothionein (MT)-1 and MT-2 mRNAs were used to compare MT gene copy numbers, zinc-induced MT mRNA levels, and uninduced MT mRNA levels in cadmium-resistant (Cdr) Chinese hamster ovary cell lines. Quantitative hybridization analyses determined that the MT-1 and MT-2 genes are each present at approximately single-copy levels in the genome of cell line Cdr2C10 and are coordinately amplified approximately 7, 3, and 12 times over the Cdr2C10 value in the genomes of cell lines Cdr20F4, Cdr30F9, and Cdr200T1, respectively. The maximum zinc-induced MT-1 mRNA concentrations in cell lines Cdr20F4, Cdr30F9, and Cdr200T1 were equal to 1, 3, and 15 times that measured in Cdr2C10, respectively. Similarly, the maximum zinc-induced MT-2 mRNA concentrations were equal to 1, 3, and 14 times that measured in Cdr2C10, respectively, and in each instance they were 90 to 150 times greater than their respective concentrations in uninduced cells. Thus, relative MT gene numbers are closely correlated with both zinc-induced and uninduced MT mRNA levels in Cdr2C10, Cdr30F9, and Cdr200T1, but not in Cdr20F4. Each of the latter two lines possesses structurally altered chromosomes whose breakpoints are near the MT locus. Nonetheless, the ratio of the levels of MT-1 to MT-2 mRNAs was constant in each of the four cell lines, including Cdr20F4. These results demonstrate that MT-1 and MT-2 mRNAs are induced coordinately in each Cdr cell line. Therefore, the coordination of the induction of MT-1 and MT-2 mRNA is independent of MT gene amplification, MT gene rearrangement, and the relative inducibilities of amplified MT genes. However, MT mRNA and protein levels each indicate that MT-1 and MT-2 expression is non-coordinate in uninduced cells. Thus, regulation of MT expression may involve two different mechanisms which are differentially operative in induced and uninduced cells.

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A Role for Topoisomerase I in Fusarium graminearum and F. culmorum Pathogenesis and Sporulation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-5-0566 ◽

2010 ◽

Vol 23 (5) ◽

pp. 566-577 ◽

Cited By ~ 29

Author(s):

Thomas K. Baldwin ◽

Martin Urban ◽

Neil Brown ◽

Kim E. Hammond-Kosack

Keyword(s):

Fusarium Graminearum ◽

Topoisomerase I ◽

Insertional Mutagenesis ◽

Hyphal Growth ◽

Single Copy ◽

Forward Genetics ◽

Host Responses ◽

Trichothecene Mycotoxin ◽

Gene Coding

Fusarium graminearum and F. culmorum are the causal agents of Fusarium ear blight (FEB) in wheat. A forward genetics approach was taken to discover novel pathogenicity genes in the genome of F. graminearum. A library of transformants created by random plasmid insertional mutagenesis was screened on wheat ears for virulence defects. Plasmid rescue on one of the reduced-virulence mutants revealed a single-copy plasmid insertion in the gene coding for the DNA interacting enzyme, topoisomerase I. Targeted topoisomerase I gene-deletion mutants were created in strains of both F. graminearum and F. culmorum. The top1 mutants of both species exhibited greatly reduced virulence in wheat ear infection assays (GO:0009405 and GO:0044145). Detailed microscopy analyses revealed that top1 hyphal growth was restricted to palea tissue whereas host responses were discernable 1,000 μm further away in the rachis node. Asexual sporulation was reduced in the F. graminearum mutants and was absent from the F. culmorum mutants. The F. graminearum mutant did not develop sexual spores when subjected to an in vitro perithecia production assay. During in vitro growth, the top1 mutants of both species were still able to produce the trichothecene mycotoxin, deoxynivalenol.

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Disruption of single-copy genes encoding acidic ribosomal proteins in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2182 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2182-2190 ◽

Cited By ~ 35

Author(s):

M Remacha ◽

C Santos ◽

J P Ballesta

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Proteins ◽

Minimal Medium ◽

Single Copy ◽

Diploid Strain ◽

Tetrad Analysis ◽

Genes Encoding ◽

Acidic Proteins ◽

Dna Fragment ◽

His3 Gene

Using the cloned genes coding for the ribosomal acidic proteins L44 and L45, constructions were made which deleted part of the coding sequence and inserted a DNA fragment at that site carrying either the URA3 or HIS3 gene. By gene disruption techniques with linearized DNA from these constructions, strains of Saccharomyces cerevisiae were obtained which lacked a functional gene for either protein L44 or protein L45. The disrupted genes in the transformants were characterized by Southern blots. The absence of the proteins was verified by electrofocusing and immunological techniques, but a compensating increase of the other acidic ribosomal proteins was not detected. The mutant lacking L44 grew at a rate identical to the parental strain in complex as well as in minimal medium. The L45-disrupted strain also grew well in both media but at a slower rate than the parental culture. A diploid strain was obtained by crossing both transformants, and by tetrad analysis it was shown that the double transformant lacking both genes is not viable. These results indicated that proteins L44 and L45 are independently dispensable for cell growth and that the ribosome is functional in the absence of either of them.

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Endogenous RNA interference is driven by copy number

eLife ◽

10.7554/elife.01581 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 17

Author(s):

Cristina Cruz ◽

Jonathan Houseley

Keyword(s):

Rna Interference ◽

Transposable Elements ◽

Copy Number ◽

Defense Mechanism ◽

Gene Copy Number ◽

Single Copy ◽

Gene Copy ◽

Protein Coding ◽

Endogenous Loci ◽

Pervasive Transcription

A plethora of non-protein coding RNAs are produced throughout eukaryotic genomes, many of which are transcribed antisense to protein-coding genes and could potentially instigate RNA interference (RNAi) responses. Here we have used a synthetic RNAi system to show that gene copy number is a key factor controlling RNAi for transcripts from endogenous loci, since transcripts from multi-copy loci form double stranded RNA more efficiently than transcripts from equivalently expressed single-copy loci. Selectivity towards transcripts from high-copy DNA is therefore an emergent property of a minimal RNAi system. The ability of RNAi to selectively degrade transcripts from high-copy loci would allow suppression of newly emerging transposable elements, but such a surveillance system requires transcription. We show that low-level genome-wide pervasive transcription is sufficient to instigate RNAi, and propose that pervasive transcription is part of a defense mechanism capable of directing a sequence-independent RNAi response against transposable elements amplifying within the genome.

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437 DETERMINING GENE COPY NUMBER IN TRANSFECTED CAPRINE FIBROBLAST CELLS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab437 ◽

2010 ◽

Vol 22 (1) ◽

pp. 375

Author(s):

J. A. Wilson ◽

R. A. Godke ◽

K. R. Bondioli

Keyword(s):

Dna Sequences ◽

Copy Number ◽

Genomic Dna ◽

Gene Copy Number ◽

Single Copy ◽

Gene Copy ◽

Transgene Copy Number ◽

Transgenic Organisms ◽

Plasmid Construct ◽

The Mean

Transgene expression in stably transgenic organisms is affected by many factors, including the copy number of the transgene in the genome and by interactions between the transgene and flanking DNA sequences. Very high transgene copy number has also been shown to affect genetic stability in transgenic plants and animals. Two commonly used methods for transfecting cells prior to their use in nuclear transfer (NT) are liposome-mediated transfection and electroporation. Little is known about the transgene copy number or variability of the copy number with these techniques. The objective of this study was to determine transgene copy number after liposome-mediated transfection and electroporation. The mean transgene copy number and variability between individual integration events have been determined. Q-PCR conditions were optimized for primer annealing temperature and concentration when amplifying a region of a plasmid expressing green fluorescent protein (GFP) under the control of the human elongation factor (hEF) promoter (hEFGFP) used for transfection. The quantitative nature of the Q-PCR reaction was confirmed by amplifying 10-fold dilutions of the plasmid and plotting the threshold cycle (CT) value against the log of the plasmid concentration. A correlation coefficient of 1.00 and a calculated PCR efficiency of 93.3% were obtained from this analysis. Caprine fibroblasts were transfected by electroporation with 20 μg of DNA or FuGENE® HD (Roche, Nutley, NJ, USA) reagent with 6 μg of DNA using either a circular or linearized hEFGFP plasmid. Transformed cells were plated at low density in medium containing Geneticin® (Gibco, Grand Island, NY USA). After 10 days of culture, single-cell colonies were isolated and expanded. When cultures reached 1 to 2 million cells, genomic DNA was isolated. Transgene copy number was determined by amplifying genomic DNA from individual clones representing 1 × 105 cells with Q-PCR. Transgene copy number was calculated from a standard curve of the transgene plasmid. The mean transgene copy number for electroporation circular was 2.7 ± 0.75 (n = 32 colonies) and 1.3 ± 0.65 (n = 19) when using a linear DNA construct. FuGENE HD using a circular plasmid construct generated a mean gene copy number of 0.5 ± 0.11 (n = 14) and 0.64 ± 0.13 (n = 16) for the linear plasmid construct. One-way ANOVA followed by multiple pair-wise comparisons using Tukey’s method showed significant differences when comparing electroporation circular to all other treatments. However, there were no differences when comparing electroporation linear, FuGENE HD circular, and FuGENE HD linear to each other. Because the calculated mean copy number for transfection with FuGENE HD was consistently less than 1, it is assumed that these colonies consisted predominantly of single-copy integrations. Our results indicate that the transfection method can affect gene copy number. Electroporation resulted in multiple but few copies whereas Fugene HD resulted in predominantly single-copy integrations. The probability of transgene mutation with single-copy integration suggests that electroporation is preferable forproducing transgenic animals by NT.

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Disruption of single-copy genes encoding acidic ribosomal proteins in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2182-2190.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2182-2190

Author(s):

M Remacha ◽

C Santos ◽

J P Ballesta

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Proteins ◽

Minimal Medium ◽

Single Copy ◽

Diploid Strain ◽

Tetrad Analysis ◽

Genes Encoding ◽

Acidic Proteins ◽

Dna Fragment ◽

His3 Gene

Using the cloned genes coding for the ribosomal acidic proteins L44 and L45, constructions were made which deleted part of the coding sequence and inserted a DNA fragment at that site carrying either the URA3 or HIS3 gene. By gene disruption techniques with linearized DNA from these constructions, strains of Saccharomyces cerevisiae were obtained which lacked a functional gene for either protein L44 or protein L45. The disrupted genes in the transformants were characterized by Southern blots. The absence of the proteins was verified by electrofocusing and immunological techniques, but a compensating increase of the other acidic ribosomal proteins was not detected. The mutant lacking L44 grew at a rate identical to the parental strain in complex as well as in minimal medium. The L45-disrupted strain also grew well in both media but at a slower rate than the parental culture. A diploid strain was obtained by crossing both transformants, and by tetrad analysis it was shown that the double transformant lacking both genes is not viable. These results indicated that proteins L44 and L45 are independently dispensable for cell growth and that the ribosome is functional in the absence of either of them.

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Dictyostelium discoideum contains two profilin isoforms that differ in structure and function

Journal of Cell Science ◽

10.1242/jcs.100.3.481 ◽

1991 ◽

Vol 100 (3) ◽

pp. 481-489 ◽

Cited By ~ 2

Author(s):

M. Haugwitz ◽

A.A. Noegel ◽

D. Rieger ◽

F. Lottspeich ◽

M. Schleicher

Keyword(s):

Amino Acid ◽

Cdna Library ◽

Dictyostelium Discoideum ◽

Actin Polymerization ◽

Critical Concentration ◽

Exchange Chromatography ◽

Amino Acid Sequences ◽

Actin Binding ◽

Gene Coding ◽

And Function

Two profilin isoforms (profilins I and II) have been purified from Dictyostelium discoideum, using affinity chromatography on a poly(L-proline) matrix; the isoforms could be separated by cation-exchange chromatography on a FPLC system. The gene coding for profilin I was cloned from a lambda gt11 cDNA library using a profilin I-specific monoclonal antibody. The profilin II cDNA was isolated by probing the cDNA library with an oligonucleotide deduced from the N-terminal amino acid sequence of profilin II, which has an open N terminus in contrast to profilin I. The deduced amino acid sequences of both genes show that profilin I in comparison to profilin II is slightly larger (13,064 Da vs 12,729 Da), has a more acidic isoelectric point (calc. pI 6.62 vs 7.26) and shares with profilin II 68 identical residues out of 126 amino acids. Although both profilins contain a conserved lysine residue in the putative actin-binding region and can be crosslinked covalently to G-actin, the crosslinking efficiency of profilin II to actin is substantially higher than that of profilin I. These data are in agreement with studies on the functional properties of the profilin isoforms. In most preparations profilin II was more efficient in delaying the onset of elongation during the course of actin polymerization and caused a higher critical concentration for actin polymerization than profilin I, probably due to the slightly increased affinity of profilin II for D. discoideum G-actin (approx. Kd 1.8 × 10(−6) M) as compared to that of profilin I (approx. Kd 5.1 × 10(−6) M).(ABSTRACT TRUNCATED AT 250 WORDS)

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Novel Isolate of Bacillus thuringiensis subsp. thuringiensis That Produces a Quasicuboidal Crystal of Cry1Ab21 Toxic to Larvae of Trichoplusia ni

Applied and Environmental Microbiology ◽

10.1128/aem.01955-07 ◽

2007 ◽

Vol 74 (4) ◽

pp. 923-930 ◽

Cited By ~ 24

Author(s):

Izabela Swiecicka ◽

Dennis K. Bideshi ◽

Brian A. Federici

Keyword(s):

Bacillus Thuringiensis ◽

Trichoplusia Ni ◽

Single Copy ◽

Cabbage Looper ◽

Beet Armyworm ◽

Vegetable Crops ◽

Gene Coding ◽

Novel Variant ◽

Northeastern Poland ◽

Midgut Lumen

ABSTRACT A new isolate (IS5056) of Bacillus thuringiensis subsp. thuringiensis that produces a novel variant of Cry1Ab, Cry1Ab21, was isolated from soil collected in northeastern Poland. Cry1Ab21 was composed of 1,155 amino acids and had a molecular mass of 130.5 kDa, and a single copy of the gene coding for this endotoxin was located on a ∼75-kbp plasmid. When synthesized by the wild-type strain, Cry1Ab21 produced a unique, irregular, bipyramidal crystal whose long and short axes were both approximately 1 μm long, which gave it a cuboidal appearance in wet mount preparations. In diet incorporation bioassays, the 50% lethal concentrations of the crystal-spore complex were 16.9 and 29.7 μg ml−1 for second- and fourth-instar larvae of the cabbage looper, Trichoplusia ni, respectively, but the isolate was essentially nontoxic to larvae of the beet armyworm, Spodoptera exigua. A bioassay of autoclaved spore-crystal preparations showed no evidence of β-exotoxin activity, indicating that toxicity was due primarily to Cry1Ab21. Studies of the pathogenesis of isolate IS5056 in second-instar larvae of T. ni showed that after larval death the bacterium colonized and subsequently sporulated extensively throughout the cadaver, suggesting that other bacteria inhabiting the midgut lumen played little if any role in mortality. As T. ni is among the most destructive pests of vegetable crops in North America and has developed resistance to B. thuringiensis, this new isolate may have applied value.

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