Evidence that the functional beta-actin gene is single copy in most mice and is associated with 5' sequences capable of conferring serum- and cycloheximide-dependent regulation.

Elder, P K; French, C L; Subramaniam, M; Schmidt, Lucy J.; Getz, Michael J.

doi:10.1128/mcb.8.1.480

Cited by 60 publications

(36 citation statements)

References 20 publications

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“…The 5Ј primer includes an EcoRI restriction site (underlined) followed by ␤-actin intron 1 sequence (this sequence was kindly provided by Dr. Michael Getz, Mayo Clinic, Rochester, MN). The 3Ј primer includes the BamHI restriction site (underlined) followed by ␤-actin exon 2 sequence (44). Following amplification, the DNA fragments were gel-purified and ligated into pBluescript KS ϩ .…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Processing of Glucose-6-phosphate Dehydrogenase mRNA by Nutritional Status

Amir-Ahmady

Salati

2001

Journal of Biological Chemistry

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Expression of glucose-6-phosphate dehydrogenase (G6PD) gene during starvation and refeeding is regulated by a posttranscriptional mechanism occurring in the nucleus. The amount of G6PD mRNA at different stages of processing was measured in RNA isolated from the nuclear matrix fraction of mouse liver. This nuclear fraction contains nascent transcripts and RNA undergoing processing. Using a ribonuclease protection assay with probes that cross an exon-intron boundary in the G6PD transcript, the abundance of mRNAs that contain the intron (unspliced) and without the intron (spliced) was measured. Refeeding resulted in 6-and 8-fold increases in abundance of G6PD unspliced and spliced RNA, respectively, in the nuclear matrix fraction. However, the amount of G6PD unspliced RNA was at most 15% of the amount of spliced RNA. During refeeding, G6PD spliced RNA accumulated at a rate significantly greater than unspliced RNA. Further, the amount of partially spliced RNA exceeded the amount of unspliced RNA indicating that the enhanced accumulation occurs early in processing. Starvation and refeeding did not regulate either the rate of polyadenylation or the length of the poly(A) tail. Thus, the G6PD gene is regulated during refeeding by enhanced efficiency of splicing of its RNA, and this processing protects the mRNA from decay, a novel mechanism for nutritional regulation of gene expression.Glucose-6-phosphate dehydrogenase (G6PD, 1 EC 1.1.1.49) is the rate-limiting enzyme of the pentose phosphate pathway. All cells contain G6PD activity; however, nutritional and hormonal factors only regulate the expression of the enzyme in liver and adipose tissue (1-3). Regulation of G6PD activity in these tissues is because it plays a critical role in the de novo synthesis of fatty acids by providing 50 -75% of the NADPH for the fatty acid synthase reaction (4). Thus, G6PD is a member of the lipogenic enzyme family. The activities of the lipogenic enzymes are coordinately regulated so that flux of substrate through the fatty acid biosynthetic pathway is high when animals consume a diet rich in carbohydrate and flux is decreased by starvation or the addition of polyunsaturated fat to the diet (reviewed in Ref. 5). However, the molecular mechanisms causing these changes in flux vary considerably between enzymes.The enzymes of the fatty acid biosynthetic pathway are regulated at both transcriptional and posttranscriptional steps. Fatty acid synthase (6 -8), acetyl-CoA carboxylase (9, 10), stearoyl-CoA desaturase (11), and ATP-citrate lyase (12) undergo large changes in their transcriptional rates in response to dietary manipulations. Regulation of malic enzyme expression during a chow to fat-free diet transition occurs by changes in mRNA stability in the cytoplasm (13). In addition, cytoplasmic mRNA stability is also involved in the regulation of stearoylCoA desaturase expression by fatty acids in yeast (14) and in adipocytes (15). Posttranscriptional regulation is the primary mechanism involved in the nutritional regulation of G6PD expressi...

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Section: Methodsmentioning

confidence: 99%

Regulation of the Processing of Glucose-6-phosphate Dehydrogenase mRNA by Nutritional Status

Amir-Ahmady

Salati

2001

Journal of Biological Chemistry

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“…However, expression of putative ' stable ' housekeeping genes [e.g. those coding for dihydrofolate reductase (DHFR) or β-actin] may vary as much as that of the target gene [6,7].…”

Section: Introductionmentioning

confidence: 99%

Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR

Zhang

Byrne

1999

Biochemical Journal

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Quantitative competitive reverse-transcriptase PCR is the most sensitive method for studying gene expression. To investigate whether the accuracy of the calculated target mRNA copy number is affected by the cDNA priming process, we utilized primers of different lengths, concentrations and primer sequences to prime cDNA synthesis reactions. Our results show a ≈ 19-fold increase in the calculated mRNA copy number from cDNA synthesis reactions primed with random hexamers (P< 0.001, n = 4), and a ≈ 4-fold increase in copy number with a specific hexamer (P< 0.001, n = 4) compared with that obtained with a 22-mer-sequence-specific primer. The increase in calculated mRNA copy number obtained by priming cDNA synthesis with the shorter specific and non-specific primers could be explained largely by the synthesis of truncated standard cDNA molecules lacking a requisite binding site for amplification with PCR primers. Since these truncated standard cDNA molecules could not be amplified and standard RNA is used to quantify target mRNA copy number, this phenomenon resulted in overestimation of target mRNA copy number. In conclusion, accurate determination of target mRNA copy number is most likely if a long specific antisense primer is used to prime cDNA synthesis.

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“…An 11.0 kb EcoRI fragment containing all six exons of the cytoplasmic b-actin gene (Elder et al, 1988) was isolated from a genomic clone (provided by the laboratory of Dr Michael Getz) and used for constructing a targeting vector cassette ('actin cassette') in several steps. The final construct (Figure 1a) consisted of 5 0 and 3 0 regions of actin locus homology flanking an IRES element, a promotorless neo gene, and a cDNA encoding PVMT.…”

Section: Construction Of Targeting Vectorsmentioning

confidence: 99%

‘Designer’ tumors in mice

et al. 2003

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We have developed and tested successfully a general method based on Cre-mediated recombination that can be used for ubiquitous or tissue-specific expression of protein products, including tumor-inducing oncoproteins. Depending on the specificity of a chosen promoter driving cre expression, tumors develop by design in bitransgenic mouse progeny derived by crossing Cre-producing mice with partners carrying a dormant oncogenic transgene (targeted into the 3 0 noncoding region of the cytoplasmic b-actin locus) that becomes functional after excision of a 'floxed' DNA segment. To provide proof-of-principle, we have used as models transgenes encoding the polyomavirus middle T antigen (PVMT) and the T antigens of the SV40 early region (SVER). Cre-dependent activation of widespread SVER expression resulted in hyperplasias or invasive tumors affecting particular visceral smooth muscles, whereas Cre-dependent, mammary gland-specific expression of PVMT-induced adenocarcinomas, according to plan. Unexpectedly, we also encountered spontaneous (Cre-independent) oncogene expression occurring as a rare event, which simulates the initiation of sporadic tumors and leads to PVMT-induced hemangiomas and mammary carcinomas or SVER-induced disseminated sarcomas, thus, revealing particular tissue susceptibilities to the actions of these oncoproteins.

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Evidence that the functional beta-actin gene is single copy in most mice and is associated with 5' sequences capable of conferring serum- and cycloheximide-dependent regulation.

Cited by 60 publications

References 20 publications

Regulation of the Processing of Glucose-6-phosphate Dehydrogenase mRNA by Nutritional Status

Regulation of the Processing of Glucose-6-phosphate Dehydrogenase mRNA by Nutritional Status

Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR

‘Designer’ tumors in mice

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