Identification of the Genomic Region Which Complements a Temperature-Sensitive, High-CO2 Requiring Mutant of the Cyanobacterium, Synechococcus PCC7942

We have studied the regulation of expression of the asparagine synthetase (AS) gene in ts11 cells, a mutant of BHK hamster cells which encodes a temperature-sensitive AS and therefore does not produce endogenous asparagine at 39.5 degrees C. Incubation of ts11 cells at the nonpermissive temperature drastically increases the level of AS mRNA, and the stimulation of AS mRNA expression is effectively suppressed by the addition of asparagine to the medium. We show here that regulation of AS gene expression involves cis-acting elements which are contained in the mRNA as well as in the 5' genomic region. When a plasmid containing the human AS cDNA under the control of the human AS promoter region was stably transfected into ts11 cells, the expression of human AS RNAs was regulated as that of the endogenous hamster transcripts, indicating that this construct contained all cis elements necessary for regulation. Expression of the AS cDNA in ts11 cells under the control of a constitutive foreign promoter was also regulated by the concentration of asparagine, and this regulation required translation. When we introduced by mutagenesis a number of stop codons in the AS cDNA, the mutant mRNAs with short open reading frames were expressed at low levels that were not increased by asparagine deprivation. Inhibition of protein and RNA synthesis also prevented down-regulation of AS mRNA levels by high concentrations of asparagine. In a parallel series of experiments, we showed that an AS DNA fragment including the promoter and first exon can also regulate RNA expression in response to asparagine concentration. Furthermore, similar increases in the levels of AS RNAs are produced not only by asparagine deprivation in ts11 cells but also by deprivation of human and wild-type BHK cells of leucine, isoleucine, or glutamine. Thus, regulation of AS gene expression is a response to amino acid starvation through mechanisms which appear to involve both changes in RNA stability and change in the rates of transcription initiation or elongation.

Download Full-text

Isolation and Characterization of High CO2-Requiring-Mutants of the Cyanobacterium Synechococcus PCC7942

PLANT PHYSIOLOGY ◽

10.1104/pp.91.2.514 ◽

1989 ◽

Vol 91 (2) ◽

pp. 514-525 ◽

Cited By ~ 113

Author(s):

G. D. Price ◽

M. R. Badger

Keyword(s):

High Co2 ◽

Isolation And Characterization ◽

Synechococcus Pcc7942

Download Full-text

Expression of Human Carbonic Anhydrase in the Cyanobacterium Synechococcus PCC7942 Creates a High CO2-Requiring Phenotype

PLANT PHYSIOLOGY ◽

10.1104/pp.91.2.505 ◽

1989 ◽

Vol 91 (2) ◽

pp. 505-513 ◽

Cited By ~ 145

Author(s):

G. D. Price ◽

M. R. Badger

Keyword(s):

Carbonic Anhydrase ◽

High Co2 ◽

Synechococcus Pcc7942 ◽

Human Carbonic Anhydrase

Download Full-text

Ribosomal S27a Coding Sequences Upstream of Ubiquitin Coding Sequences in the Genome of a Pestivirus

Journal of Virology ◽

10.1128/jvi.72.11.8697-8704.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8697-8704 ◽

Cited By ~ 25

Author(s):

Paul Becher ◽

Michaela Orlich ◽

Heinz-Jürgen Thiel

Keyword(s):

Molecular Cloning ◽

Genomic Region ◽

Nucleotide Sequencing ◽

Temperature Sensitive ◽

Nonstructural Proteins ◽

Transient Expression Assay ◽

Sequence Comparisons ◽

Coding Sequences ◽

Diarrhea Virus ◽

Processing Signal

ABSTRACT Molecular characterization of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP Rit, a temperature-sensitive strain widely used for vaccination, revealed that the viral genomic RNA is about 15.2 kb long, which is about 2.9 kb longer than the one of noncytopathogenic (noncp) BVDV strains. Molecular cloning and nucleotide sequencing of parts of the genome resulted in the identification of a duplication of the genomic region encoding nonstructural proteins NS3, NS4A, and part of NS4B. In addition, a nonviral sequence was found directly upstream of the second copy of the NS3 gene. The 3′ part of this inserted sequence encodes an N-terminally truncated ubiquitin monomer. This is remarkable since all described cp BVDV strains with ubiquitin coding sequences contain at least one complete ubiquitin monomer. The 5′ region of the nonviral sequence did not show any homology to cellular sequences identified thus far in cp BVDV strains. Databank searches revealed that this second cellular insertion encodes part of ribosomal protein S27a. Further analyses included molecular cloning and nucleotide sequencing of the cellular recombination partner. Sequence comparisons strongly suggest that the S27a and the ubiquitin coding sequences found in the genome of CP Rit were both derived from a bovine mRNA encoding a hybrid protein with the structure NH2-ubiquitin-S27a-COOH. Polyprotein processing in the genomic region encoding the N-terminal part of NS4B, the two cellular insertions, and NS3 was studied by a transient-expression assay. The respective analyses showed that the S27a-derived polypeptide, together with the truncated ubiquitin, served as processing signal to yield NS3, whereas the truncated ubiquitin alone was not capable of mediating the cleavage. Since the expression of NS3 is strictly correlated with the cp phenotype of BVDV, the altered genome organization leading to expression of NS3 most probably represents the genetic basis of cytopathogenicity of CP Rit.

Download Full-text

Regulation of asparagine synthetase gene expression by amino acid starvation.

Molecular and Cellular Biology ◽

10.1128/mcb.11.12.6059 ◽

1991 ◽

Vol 11 (12) ◽

pp. 6059-6066 ◽

Cited By ~ 63

Author(s):

S S Gong ◽

L Guerrini ◽

C Basilico

Keyword(s):

Gene Expression ◽

Amino Acid ◽

Transcription Initiation ◽

Rna Stability ◽

Genomic Region ◽

Amino Acid Starvation ◽

Asparagine Synthetase ◽

Temperature Sensitive ◽

Mrna Levels ◽

Regulation Of Expression

We have studied the regulation of expression of the asparagine synthetase (AS) gene in ts11 cells, a mutant of BHK hamster cells which encodes a temperature-sensitive AS and therefore does not produce endogenous asparagine at 39.5 degrees C. Incubation of ts11 cells at the nonpermissive temperature drastically increases the level of AS mRNA, and the stimulation of AS mRNA expression is effectively suppressed by the addition of asparagine to the medium. We show here that regulation of AS gene expression involves cis-acting elements which are contained in the mRNA as well as in the 5' genomic region. When a plasmid containing the human AS cDNA under the control of the human AS promoter region was stably transfected into ts11 cells, the expression of human AS RNAs was regulated as that of the endogenous hamster transcripts, indicating that this construct contained all cis elements necessary for regulation. Expression of the AS cDNA in ts11 cells under the control of a constitutive foreign promoter was also regulated by the concentration of asparagine, and this regulation required translation. When we introduced by mutagenesis a number of stop codons in the AS cDNA, the mutant mRNAs with short open reading frames were expressed at low levels that were not increased by asparagine deprivation. Inhibition of protein and RNA synthesis also prevented down-regulation of AS mRNA levels by high concentrations of asparagine. In a parallel series of experiments, we showed that an AS DNA fragment including the promoter and first exon can also regulate RNA expression in response to asparagine concentration. Furthermore, similar increases in the levels of AS RNAs are produced not only by asparagine deprivation in ts11 cells but also by deprivation of human and wild-type BHK cells of leucine, isoleucine, or glutamine. Thus, regulation of AS gene expression is a response to amino acid starvation through mechanisms which appear to involve both changes in RNA stability and change in the rates of transcription initiation or elongation.

Download Full-text

Fine Mapping and Identification of BnaC06.FtsH1, a Lethal Gene That Regulates the PSII Repair Cycle in Brassica napus

International Journal of Molecular Sciences ◽

10.3390/ijms22042087 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2087

Author(s):

Kai Xu ◽

Yujin Wu ◽

Jurong Song ◽

Kaining Hu ◽

Zengxiang Wu ◽

...

Keyword(s):

Brassica Napus ◽

Biomass Accumulation ◽

Genomic Region ◽

Functional Verification ◽

Homologous Gene ◽

Temperature Sensitive ◽

Lethal Gene ◽

Lethal Mutant ◽

Recessive Genes ◽

Repair Cycle

Photosystem II (PSII) is an important component of the chloroplast. The PSII repair cycle is crucial for the relief of photoinhibition and may be advantageous when improving stress resistance and photosynthetic efficiency. Lethal genes are widely used in the efficiency detection and method improvement of gene editing. In the present study, we identified the naturally occurring lethal mutant 7-521Y with etiolated cotyledons in Brassica napus, controlled by double-recessive genes (named cyd1 and cyd2). By combining whole-genome resequencing and map-based cloning, CYD1 was fine-mapped to a 29 kb genomic region using 15,167 etiolated individuals. Through cosegregation analysis and functional verification of the transgene, BnaC06.FtsH1 was determined to be the target gene; it encodes an filamentation temperature sensitive protein H 1 (FtsH1) hydrolase that degrades damaged PSII D1 in Arabidopsis thaliana. The expression of BnaC06.FtsH1 was high in the cotyledons, leaves, and flowers of B. napus, and localized in the chloroplasts. In addition, the expression of EngA (upstream regulation gene of FtsH) increased and D1 decreased in 7-521Y. Double mutants of FtsH1 and FtsH5 were lethal in A. thaliana. Through phylogenetic analysis, the loss of FtsH5 was identified in Brassica, and the remaining FtsH1 was required for PSII repair cycle. CYD2 may be a homologous gene of FtsH1 on chromosome A07 of B. napus. Our study provides new insights into lethal mutants, the findings may help improve the efficiency of the PSII repair cycle and biomass accumulation in oilseed rape.

Download Full-text

Oncogenesis and the Lucké Virus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100100743 ◽

1968 ◽

Vol 26 ◽

pp. 200-201

Author(s):

A. E. Vatter ◽

J. Zambernard

Keyword(s):

Rna Viruses ◽

Vegetative State ◽

Temperature Sensitive ◽

Structural Level ◽

Oncogenic Viruses ◽

Dna Viruses ◽

Leopard Frogs ◽

Renal Adenocarcinoma ◽

Induced Tumors ◽

Northern Leopard Frogs

Oncogenic viruses, like viruses in general, can be divided into two classes, those that contain deoxyribonucleic acid (DNA) and those that contain ribonucleic acid (RNA). The RNA viruses have been recovered readily from the tumors which they cause whereas, the DNA-virus induced tumors have not yielded the virus. Since DNA viruses cannot be recovered, the bulk of present day investigations have been concerned with RNA viruses.The Lucké renal adenocarcinoma is a spontaneous tumor which occurs in northern leopard frogs (Rana pipiens) and has received increased attention in recent years because of its probable viral etiology. This hypothesis was first advanced by Lucké after he observed intranuclear inclusions in some of the tumor cells. Tumors with inclusions were examined at the fine structural level by Fawcett who showed that they contained immature and mature virus˗like particles.The use of this system in the study of oncogenic tumors offers several unique features, the virus has been shown to contain DNA and it can be recovered from the tumor, also, it is temperature sensitive. This latter feature is of importance because the virus can be transformed from a latent to a vegetative state by lowering or elevating the environmental temperature.

Download Full-text