Rat prostatic binding protein: the complete sequence of the C2 gene and its flanking regions

Delaey, Bernard; Dirckx, Luc; Decourt, Jean-Luc; Claessens, Frank; Peeters, Benjamin; Rombauts, Wilfried

doi:10.1093/nar/15.4.1627

Cited by 13 publications

(5 citation statements)

References 41 publications

(29 reference statements)

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“…Similar homopurinehomopyrimidine tracts have been found within or proximal to other vertebrate genes (e.g. 51,52) and they frequently are hypersensitive to single strand-specific nucleases (52,53). At present, we cannot discriminate whether the decrease in activity is due to rapid degradation of the pN-1022Lucy construct after transfection, or whether it represents a natural silencing mechanism for the NF-M gene.…”

Section: Discussionmentioning

confidence: 78%

Isolation of the chicken middle-molecular weight neurofilament (NF-M) gene and characterization of its promoter

et al. 1990

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We have isolated and sequenced genomic DNA clones covering the coding region of the chicken mid-size neurofilament (NF-M) gene and greater than 1 kb of its 5' upstream region. The NF-M gene contains two introns which both are located within the highly conserved C-terminal region of the rod domain. The 5' end of the corresponding mRNA was assigned to a G residue 40 nucleotides upstream of the translation start site and in appropriate distance from a potential TATA box. To functionally analyze the NF-M promoter, constructs carrying 112, 222, and 1026 nucleotides of the 5' upstream region in front of a luciferase reporter gene were tested for their capability to direct luciferase expression after transient transfection into various cell lines. Significant luciferase activity was recorded both in rat phaeochromocytoma (PC12) cells and murine fibroblasts. In PC12 cells, in which neurite outgrowth is induced by nerve growth factor (NGF), expression was stimulated up to 13-fold within 3 days of NGF treatment. This closely resembles expression of the endogenous NF-M gene in response to this hormone.

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Section: Discussionmentioning

confidence: 78%

Isolation of the chicken middle-molecular weight neurofilament (NF-M) gene and characterization of its promoter

et al. 1990

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“…Such poly(T) tracts may represent areas of unusual chromatin structure. Homopurine and homopyrimidine tracts have been found to be sensitive to S1 nuclease digestion, presumably because of their ability to alter DNA conformation (29).…”

Section: Discussionmentioning

confidence: 99%

Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

Reid

Gregg

Smithies

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/ thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from theHPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene.Mouse embryonic stem (ES) cells are being used with increasing frequency for constructing mutations in vitro by homologous recombination (gene targeting) that can be transferred into the mouse germ-line after blastocyst injection (1)(2)(3). Such experiments are dependent to a large extent on the availability of selectable marker genes that can be expressed in mouse ES cells. To date, two such marker genes have been used: neomycin phosphotransferase (NEO) and thymidine kinase (TK) (4-6). Cells expressing the NEO gene can be selected for by their growth in medium containing G418. Conversely, cells expressing the Herpes simplex virus TK gene can be selected against due to their death in medium containing gancyclovir. In this report, we describe the identification of sequences necessary for the expression in ES cells of a third marker gene, hypoxanthine phosphoribosyltransferase (HPRT). This gene allows two selection methods. Cells expressing the HPRT gene can be either selected for by growth in medium containing hypoxanthine/aminopterin/ thymidine (HAT) or they can be selected against by culture in medium containing 6-thioguanine (6-TG).The HPRT gene is located within a 44-kilobase (kb) region on the X chromosome in humans and rodents (7)(8)(9). It contains 9 exons, which encode a 1.3-kb mRNA. While previous studies have shown that HPRT cDNA driven by a number of different promoters can be expressed after transfection into somatic cell lines, we demonstrate in this report that similar constructs rarely yield HAT-resistant ES cell colonies. We therefore began to search for elements within the gene that influence its expression in these cells.Recent analyses of a number of genes have shown that important regulatory sequences such as transcriptional enhancers or suppressors are sometimes contained in introns (10-17). Also, several reports have suggested that the presence of an intron, even one from a foreign gene, may contribute in a general way to the level of expression of a gene...

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“…Most unexpectedly, only the Cl transcripts were de¬ tected, but no hybridizing signals were observed with probes b and c (data not shown). In contrast, however, another C2A-specific probe, TCCATA-CATTATCTTATT, complementary to the gen¬ omic sequence at position + 1882 to + 1899 in exon 2 (Delaey et al 1987), did hybridize to the C2A mRNA on the same blots. These negative results with probes b and c were probably due to the pres¬ ence of a secondary structure in the 5' end of the C2 mRNAs, preventing hybridization with these probes (see Discussion).…”

Section: C2 Genesmentioning

confidence: 86%

“…They contain the complete C2A gene within a 12 kb EcoRI fragment. Figure 16 shows the restriction enzyme map of the PstI subfragment pC2A(44) containing the total gene and its flanking sequences (Delaey et al 1987).…”

Section: C2 Genesmentioning

confidence: 99%

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The androgen-dependent rat prostatic binding protein: comparison of the sequences in the 5′ part and upstream region of the Cl and C2 genes and analysis of their transcripts

Claessens

Dirckx²,

Delaey³

et al. 1989

Journal of Molecular Endocrinology

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The complete gene encoding the polypeptide Cl of the complex androgen-controlled prostatic binding protein was isolated from a rat genomic library. A new genomic fragment (C2B) containing only the 5\ m=' \ part of a C2-related gene was also purified. The segments containing exon 1 and a large part of the adjacent sequences were analysed and compared with the corresponding region of the C2A gene which has been completely sequenced previously. The high structural similarity extending over a large part of all three genomic fragments suggests the duplication of a common ancestral gene, followed by a more recent duplication of the C2-coding region. However, since the structural similarity upstream of position \m=-\150 between C2A and C2B abruptly disappears and no transcripts specific for the C2B region can be detected in prostate RNA, we propose that at a later stage in evolution the C2B region was disrupted and inactivated. Despite the common origin and the similar regulation of the two active genes, Cl and C2A, the only obvious conserved structural element is the homopurine stretch located at position \m=-\400, although sequence motifs resembling steroid hormone response elements are present at several locations.

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Rat prostatic binding protein: the complete sequence of the C2 gene and its flanking regions

Cited by 13 publications

References 41 publications

Isolation of the chicken middle-molecular weight neurofilament (NF-M) gene and characterization of its promoter

Isolation of the chicken middle-molecular weight neurofilament (NF-M) gene and characterization of its promoter

Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

The androgen-dependent rat prostatic binding protein: comparison of the sequences in the 5′ part and upstream region of the Cl and C2 genes and analysis of their transcripts

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