scholarly journals An eleven nucleotide section of the 3′-untranslated region is required for perinuclear localization of rat metallothionein-1 mRNA

2005 ◽  
Vol 387 (2) ◽  
pp. 419-428 ◽  
Author(s):  
David NURY ◽  
Hervé CHABANON ◽  
Marilyne LEVADOUX-MARTIN ◽  
John HESKETH
Keyword(s):  
Metal Binding ◽  
Untranslated Region ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mrna Localization ◽  
Coding Region ◽  
Ovary Cells ◽  
Structural Context ◽  
Deletion Mutagenesis ◽  
Localization Signal

Localization of mRNAs provides a novel mechanism for synthesis of proteins close to their site of function. MT1 (metallothionein-1) is a small, metal-binding protein that is largely cytoplasmic but which can be found in the nucleus. The localization of rat MT1 requires the perinuclear localization of its mRNA by a mechanism dependent on the 3′-UTR (3′-untranslated region). The present study investigates the nature of this mRNA localization signal using Chinese-hamster ovary cells transfected with gene constructs in which either MT1 or the globin coding region is linked to different sequences from the MT1 3′-UTR. Deletion, mutagenesis and antisense oligonucleotide approaches indicate that nt 45–76 of the 3′-UTR, in particular nt 66–76, are required for the localization of either MT1 mRNA or chimaeric transcripts in which a β-globin coding region is linked to sequences from the MT1 3′-UTR. This section of the 3′-UTR contains a CACC repeat. Two mutations that are predicted to alter the secondary structure of this region also impair localization. Our hypothesis is that the perinuclear localization signal in MT1 mRNA is formed by a combination of the CACC repeat and its structural context.

Cloning of 11 α-tubulin gene sequences from the genome of Chinese hamster ovary cells

1985 ◽  
Vol 63 (6) ◽  
pp. 511-518 ◽  
Author(s):  
Elizabeth M. Elliott ◽  
Farida Sarangi ◽  
Graham Henderson ◽  
Victor Ling
Keyword(s):  
Chinese Hamster Ovary ◽  
Genomic Library ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Coding Region ◽  
Tubulin Gene ◽  
Ovary Cells ◽  
Tubulin Genes ◽  
Clonal Lines ◽  
Β Tubulin

We have analyzed the complex tubulin gene family in clonal lines of Chinese hamster ovary cells. There are approximately 16 α-tubulin genes and a similar multiplicity of β-tubulin genes. The α-tubulin genes are not closely linked to each other nor to the β-tubulin genes. A genomic library has been constructed in the vector λ charon 4A containing insert sizes of 13–20 kilobases. The library has been screened with both inter- and intra-species α-tubulin probes. Eleven α-tubulin clones with different restriction patterns have been isolated and characterized. At least seven of these clones contain the complete gene coding region. One clone appears to represent the transcribed α-tubulin gene II. The sequence of an intron from this gene is compared with that from an equivalent gene in the rat.

scholarly journals Identification of a Potential Receptor for Both Peptide Histidine Isoleucine and Peptide Histidine Valine

Endocrinology ◽  
2002 ◽  
Vol 143 (4) ◽  
pp. 1327-1336 ◽  
Author(s):  
Dicky Lai-Yin Tse ◽  
Ronald Ting-Kai Pang ◽  
Anderson On-Lam Wong ◽  
Siu-Ming Chan ◽  
Hubert Vaudry ◽  
...  
Keyword(s):  
Gall Bladder ◽  
Adenylate Cyclase ◽  
Chinese Hamster Ovary Cells ◽  
Receptor Gene ◽  
Chinese Hamster ◽  
Functional Expression ◽  
Coding Region ◽  
Ovary Cells ◽  
Pituitary Adenylate Cyclase ◽  
First Time

Abstract Peptide histidine isoleucine (PHI), peptide histidine valine (PHV), and vasoactive intestinal polypeptide (VIP) are cosynthesized from the same precursor and share high levels of structural similarities with overlapping biological functions. In this study, the first PHI/PHV receptor was isolated and characterized in goldfish. To study this receptor using homologous peptides, we have also characterized the goldfish prepro-PHI/VIP, and, surprisingly, a shorter transcript lacking the VIP coding region was isolated. A PHI/VIP precursor without the VIP coding sequence has never before been reported. Initial functional expression of the PHI/PHV receptor in Chinese hamster ovary cells revealed that it could be activated by human PHV [50% effective concentration (EC50): 43 nm] and to a lesser extent human PHI (EC50: 133 nm) and helodermin (EC50: 166 nm) but not fish and mammalian pituitary adenylate cyclase-activating polypeptides and VIPs. Subsequent studies indicated that, similar to the pituitary adenylate cyclase-activating polypeptide receptors (PAC1-R, VPAC1-R, and VPAC2-R), the receptor isolated in this study is able to interact with goldfish PHI and its C-terminally extended form, PHV with EC50 values 93 and 43 nm, respectively. Northern blot and RT-PCR/Southern blot analyses revealed that the PHI/VIP gene is expressed in the intestine, brain, and gall bladder and the PHI/PHV receptor gene is primarily expressed in the pituitary and to a lesser extend in the intestine and gall bladder, suggesting that PHI/PHV may play a role, notably in the regulation of pituitary function. In conclusion, our results demonstrate for the first time the existence of a PHI/PHV receptor, indicating that the functions of PHI and PHV could be mediated by their own receptor in addition to VIP receptors.

scholarly journals Expression and amplification of engineered mouse dihydrofolate reductase minigenes.

1983 ◽  
Vol 3 (2) ◽  
pp. 257-266 ◽  
Author(s):  
G F Crouse ◽  
R N McEwan ◽  
M L Pearson
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Specific Inhibitor ◽  
Cdna Clones ◽  
Coding Region ◽  
Chromosomal Dna ◽  
Lambda Phage ◽  
Ovary Cells

We constructed mouse dihydrofolate reductase (DHFR) minigenes (dhfr) that had 1.5 kilobases of 5' flanking sequences and contained either none or only one of the intervening sequences that are normally present in the coding region. They were greater than or equal to 3.2 kilobase long, about one-tenth the size of the corresponding chromosomal gene. Both of these minigenes complemented the DHFR deficiency in Chinese hamster ovary dhfr-1-cells at a high frequency after DNA-mediated gene transfer. The level of DHFR enzyme in various transfected clones varied over a 10-fold range but never was as high as in wild-type Chinese hamster ovary cells. In addition, the level of DHFR in primary transfectants did not vary directly with the copy number of the minigene, which ranged from fewer than five to several hundred per genome. The minigenes could be amplified to a level of over 2,000 copies per genome upon selection in methotrexate, a specific inhibitor of DHFR. In one case, the amplified minigenes were present in a tandem array; in two other cases, a rearranged minigene plasmid and its flanking chromosomal DNA sequence were amplified. Thus, the mouse dhfr minigenes could be transcribed, expressed, and amplified in Chinese hamster ovary cells, although the efficiency of expression was generally low. The key step in the construction of these minigenes was the generation in vivo of lambda phage recombinants by overlapping regions of homology between genomic and cDNA clones. The techniques used here for dhfr should be generally applicable to any gene, however large, and could be used to generate novel genes from members of multigene families.

Influence of metallothionein-1 localization on its function

2001 ◽  
Vol 355 (2) ◽  
pp. 473-479 ◽  
Author(s):  
Marilyne LEVADOUX-MARTIN ◽  
John E. HESKETH ◽  
John H. BEATTIE ◽  
Heather M. WALLACE
Keyword(s):  
Dna Damage ◽  
Uv Light ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Protective Role ◽  
S Phase ◽  
Coding Region ◽  
Ovary Cells ◽  
In Cells

Metallothioneins (MTs) have a major role to play in metal metabolism, and may also protect DNA against oxidative damage. MT protein has been found localized in the nucleus during S-phase. The mRNA encoding the MT-1 isoform has a perinuclear localization, and is associated with the cytoskeleton; this targeting, due to signals within the 3′-untranslated region (3′-UTR), facilitates nuclear localization of MT-1 during S-phase [Levadoux, Mahon, Beattie, Wallace and Hesketh (1999) J. Biol. Chem. 274, 34961-34966]. Using cells transfected with MT gene constructs differing in their 3′-UTRs, the role of MT protein in the nucleus has been studied. Chinese hamster ovary cells were transfected with either the full MT gene (MTMT cells) or with the MT 5′-UTR and coding region linked to the 3′-UTR of glutathione peroxidase (MTGSH cells). Cell survival following exposure to oxidative stress and chemical agents was higher in cells expressing the native MT gene than in cells where MT localization was disrupted, or in untransfected cells. Also, MTMT cells showed less DNA damage than MTGSH cells in response to either hydrogen peroxide or mutagen. After exposure to UV light or mutagen, MTMT cells showed less apoptosis than MTGSH cells, as assessed by DNA fragmentation and flow cytometry. The data indicate that the perinuclear localization of MT mRNA is important for the function of MT in a protective role against DNA damage and apoptosis induced by external stress.

Expression and amplification of engineered mouse dihydrofolate reductase minigenes

1983 ◽  
Vol 3 (2) ◽  
pp. 257-266
Author(s):  
G F Crouse ◽  
R N McEwan ◽  
M L Pearson
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Specific Inhibitor ◽  
Cdna Clones ◽  
Coding Region ◽  
Chromosomal Dna ◽  
Lambda Phage ◽  
Ovary Cells

We constructed mouse dihydrofolate reductase (DHFR) minigenes (dhfr) that had 1.5 kilobases of 5' flanking sequences and contained either none or only one of the intervening sequences that are normally present in the coding region. They were greater than or equal to 3.2 kilobase long, about one-tenth the size of the corresponding chromosomal gene. Both of these minigenes complemented the DHFR deficiency in Chinese hamster ovary dhfr-1-cells at a high frequency after DNA-mediated gene transfer. The level of DHFR enzyme in various transfected clones varied over a 10-fold range but never was as high as in wild-type Chinese hamster ovary cells. In addition, the level of DHFR in primary transfectants did not vary directly with the copy number of the minigene, which ranged from fewer than five to several hundred per genome. The minigenes could be amplified to a level of over 2,000 copies per genome upon selection in methotrexate, a specific inhibitor of DHFR. In one case, the amplified minigenes were present in a tandem array; in two other cases, a rearranged minigene plasmid and its flanking chromosomal DNA sequence were amplified. Thus, the mouse dhfr minigenes could be transcribed, expressed, and amplified in Chinese hamster ovary cells, although the efficiency of expression was generally low. The key step in the construction of these minigenes was the generation in vivo of lambda phage recombinants by overlapping regions of homology between genomic and cDNA clones. The techniques used here for dhfr should be generally applicable to any gene, however large, and could be used to generate novel genes from members of multigene families.

scholarly journals Differential expression of three alpha-tubulin genes in Chinese hamster ovary cells.

1985 ◽  
Vol 5 (1) ◽  
pp. 236-241 ◽  
Author(s):  
E M Elliott ◽  
H Okayama ◽  
F Sarangi ◽  
G Henderson ◽  
V Ling
Keyword(s):  
Chinese Hamster Ovary ◽  
Sequence Data ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Cdna Libraries ◽  
Coding Region ◽  
Ovary Cells ◽  
Alpha Tubulin ◽  
Tubulin Genes

Chinese hamster ovary cells contain a complex family of ca. 16 unique alpha-tubulin sequences and a similar multiplicity of beta sequences. To examine which members of this multigene family are expressed, we constructed cDNA libraries from two Chinese hamster ovary cell lines according to the method of H. Okayama and P. Berg (Mol. Cell. Biol. 3:280-289, 1983). Each library consisted of 5.5 X 10(5) transformants and contained a high percentage of full-length tubulin clones. Three different alpha-tubulin genes were identified by sequence analysis of the 3' noncoding regions of these tubulin clones. The relative abundance of the transcripts corresponding to the three genes was estimated by gene-specific dot blotting of 96 cDNA alpha-tubulin clones and was found to be 71, 24, and 5%. There is little homology in the 3' noncoding sequences of these genes; however, a strong interspecies homology exists in this region for two of the Chinese hamster ovary genes with the two alpha-tubulin genes previously described in other systems. The third Chinese hamster ovary gene, with an expression frequency of 24%, is unique in that its 3' noncoding region is unlike that of the other mammalian alpha-tubulin genes. In addition, limited sequence data from the coding region of this gene indicates it codes for a unique alpha-tubulin protein.

scholarly journals Conservation of promoter sequence but not complex intron splicing pattern in human and hamster genes for 3-hydroxy-3-methylglutaryl coenzyme A reductase.

1987 ◽  
Vol 7 (5) ◽  
pp. 1881-1893 ◽  
Author(s):  
K L Luskey
Keyword(s):  
Transcription Initiation ◽  
Untranslated Region ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Untranslated Regions ◽  
Hmg Coa Reductase ◽  
Ovary Cells ◽  
Splicing Pattern ◽  
Reductase Gene ◽  
Coa Reductase

Regulation of the expression of 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase is a critical step in controlling cholesterol synthesis. Previous studies in cultured Chinese hamster ovary cells have shown that HMG-CoA reductase is transcribed from a cholesterol-regulated promoter to yield a heterogeneous collection of mRNAs with 5' untranslated regions of 68 to 670 nucleotides in length. Synthesis of these molecules is initiated at multiple sites, and multiple donor sites are used to excise an intron in the 5' untranslated region. In the current paper, I report that human HMG-CoA reductase gene resembles the Chinese hamster gene in having multiple sites of transcription initiation that are subject to suppression by cholesterol. The human gene differs from the hamster gene in that a single donor splice site is used to excise the intron in the 5' untranslated region. All of the resulting RNAs have short 5' untranslated regions of 68 to 100 nucleotides. This difference in the splicing pattern of the first intron is species specific and not a peculiarity of cultured cells in that HMG-CoA reductase mRNAs from Syrian hamster livers resemble those of the cultured Chinese hamster ovary cells. Comparison of the DNA sequences of the HMG-CoA reductase promoters from three different species--humans, Syrian hamsters, and Chinese hamsters--shows a highly conserved region of 179 nucleotides that extends from 220 to 42 nucleotides upstream of the transcription initiation sites. This region is 88% identical between the human and Chinese hamster promoter. When fused to the coding region of the Escherichia coli chloramphenicol acetyltransferase gene, this highly conserved region of the reductase gene directs the cholesterol-regulated expression of chloramphenicol acetyltransferase in transfected hamster cells, further indicating the interspecies conservation of the regulatory elements.

Conservation of promoter sequence but not complex intron splicing pattern in human and hamster genes for 3-hydroxy-3-methylglutaryl coenzyme A reductase

1987 ◽  
Vol 7 (5) ◽  
pp. 1881-1893
Author(s):  
K L Luskey
Keyword(s):  
Transcription Initiation ◽  
Untranslated Region ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Untranslated Regions ◽  
Hmg Coa Reductase ◽  
Ovary Cells ◽  
Splicing Pattern ◽  
Reductase Gene ◽  
Coa Reductase

Regulation of the expression of 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase is a critical step in controlling cholesterol synthesis. Previous studies in cultured Chinese hamster ovary cells have shown that HMG-CoA reductase is transcribed from a cholesterol-regulated promoter to yield a heterogeneous collection of mRNAs with 5' untranslated regions of 68 to 670 nucleotides in length. Synthesis of these molecules is initiated at multiple sites, and multiple donor sites are used to excise an intron in the 5' untranslated region. In the current paper, I report that human HMG-CoA reductase gene resembles the Chinese hamster gene in having multiple sites of transcription initiation that are subject to suppression by cholesterol. The human gene differs from the hamster gene in that a single donor splice site is used to excise the intron in the 5' untranslated region. All of the resulting RNAs have short 5' untranslated regions of 68 to 100 nucleotides. This difference in the splicing pattern of the first intron is species specific and not a peculiarity of cultured cells in that HMG-CoA reductase mRNAs from Syrian hamster livers resemble those of the cultured Chinese hamster ovary cells. Comparison of the DNA sequences of the HMG-CoA reductase promoters from three different species--humans, Syrian hamsters, and Chinese hamsters--shows a highly conserved region of 179 nucleotides that extends from 220 to 42 nucleotides upstream of the transcription initiation sites. This region is 88% identical between the human and Chinese hamster promoter. When fused to the coding region of the Escherichia coli chloramphenicol acetyltransferase gene, this highly conserved region of the reductase gene directs the cholesterol-regulated expression of chloramphenicol acetyltransferase in transfected hamster cells, further indicating the interspecies conservation of the regulatory elements.

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