Methylation inhibits the interaction of DNA binding proteins with a potential c-abl intron regulatory element.

We have identified DNA binding proteins which interact with a sequence found in an intron of the tyrosine kinase coding portion of the murine c-abl gene. Several specific DNA: protein complexes were observed. Those complexes of approximate molecular weights 64 and 66kDa were detected when an Msp I site (CCGG) within the sequence was unmethylated, but were not observed when that site was methylated. Insertion of the intron sequence 5' to the rat somatic cytochrome C promoter and chloramphenicol acetyl transferase (CAT) sequences resulted in at least four-fold stimulation of CAT activity. These data suggest a potential role for the intron sequence in the regulation of gene expression.     

Identification of a growth hormone gene promoter repressor element and its cognate double- and single-stranded DNA-binding proteins

In previous investigations, cell fusion was found to silence either the endogenous rat growth hormone (GH) gene or a transfected rat GH gene promoter, implying that repression plays a role in regulation of this gene. To search the rat GH gene promoter for repressor sequences, a series of 5'-deleted GH-CAT constructs was analyzed by transient expression in GH3 rat pituitary cells. Deletion of either a distal region between positions -307/-244 or a proximal sequence between -169/-152 increased CAT enzymatic activity by 3-4-fold. Since the action of the proximal repressor element (PRE) at -169/-152 was serum-independent, and the element is located between two strong positive elements, the PRE and its cognate binding proteins were further analyzed. A 5-base pair sequence centered at -163 is critical for PRE repressor activity, since mutation of this sequence in GH-CAT constructs yielded 6-11-fold increases in expression in GH3 cells. Although the PRE is adjacent to the GH thyroid hormone (T3) response region, they are distinct elements, since the PRE mutation has little effect on the T3 response of GH-CAT constructs. Nuclear extracts of 10 cell lines were searched by DNA mobility shift for protein(s) binding specifically to a double-stranded PRE probe. No such protein was detected in any of four rodent pituitary cell lines or three human cell lines. However, three different rodent non-pituitary cell lines yielded a common shifted band, corresponding to a DNA sequence-specific PRE-binding protein (PREB). Similar analysis with the coding strand of the PRE detected no shifted band in any of these cell lines. However, the PRE noncoding strand yielded a common shifted band in all of the cell lines, corresponding to a ubiquitous, strand-specific, single-stranded PRE-binding protein (ssPREB). Mutation of the PRE permitted ssPREB binding to the coding strand, implying that the wild-type coding strand somehow excludes ssPREB binding. That PREB and ssPREB are distinct proteins was confirmed by the inability of their DNA binding sites to cross-compete binding of the proteins.     

Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages

Several classes of oligonucleotide antisense compounds of sequence complementary to the start of the mRNA coding sequence for chloramphenicol acetyl transferase (CAT), including methylphosphonate, alkyltriester, and phosphorothioate analogues of DNA, have been compared to "normal" phosphodiester oligonucleotides for their ability to inhibit expression of plasmid-directed CAT gene activity in CV-1 cells. CAT gene expression was inhibited when transfection with plasmid DNA containing the gene for CAT coupled to simian virus 40 regulatory sequences (pSV2CAT) or the human immunodeficiency virus enhancer (pHIVCAT) was carried out in the presence of 30 microM concentrations of analogue. For the oligo-methylphosphonate analogue, inhibition was dependent on both oligomer concentration and chain length. Analogues with phosphodiester linkages that alternated with either methylphosphonate, ethyl phosphotriester, or isopropyl phosphotriester linkages were less effective inhibitors, in that order. The phosphorothioate analogue was about two-times more potent than the oligo-methylphosphonate, which was in turn approximately twice as potent as the normal oligonucleotide.     

Characterization of the 5' flanking region of rat glucokinase gene

The rat glucokinase (GK) gene containing the first exon was isolated and its 5' flanking region was characterized by the bacterial chloramphenicol acetyltransferase (CAT) assay. A transient expression assay with a series of 5' deletion constructs (-5.5 k to -48) of GK-CAT fusion genes indicated that the 5' flanking sequence up to nucleotide -87 was sufficient for promoter activity in adult rat hepatocytes, but its activity was much weaker than that of the SV40 enhancer/promoter. Similar promoter activity was also detected in dRLh-84 hepatoma cells, which do not express glucokinase. Insulin treatment caused no change in the CAT activity of hepatocytes transfected with the fusion genes. These results suggest that the 5' flanking region of the glucokinase gene up to -5.5 k does not contain enhancer elements responsible for tissue-specific expression or insulin regulation.     

Relative contribution of promoter and intragenic sequences in the hormonal regulation of rat beta-casein transgenes

In order to identify DNA sequences responsible for the regulation beta-casein gene expression, lines of transgenic mice bearing the entire rat beta-casein gene and two rat beta-casein promoter chloramphenicol acetyltransferase (CAT) fusion genes have been established. All three transgenes have been shown previously to be regulated in a tissue- and stage specific manner. To investigate the relative contribution of promoter and intragenic sequences in the hormonal regulation of the beta-casein gene, mammary explant cultures derived from these lines of mice have now been performed, and the effects of PRL and glucocorticoids on transgene as compared with endogenous beta-casein gene expression have been quantified. After the addition of PRL to cultures performed in the presence of insulin and glucocorticoids, a 25- to 40-fold induction of endogenous mouse beta-casein mRNA was observed after 48 hr. A comparable greater than 25-fold induction of transgene expression after PRL addition was observed in explant cultures derived from a line of mice expressing the entire rat beta-casein gene. In contrast, PRL addition elicited only a 1- to 4.5-fold increase in CAT activity in cultures derived from two lines of mice bearing casein-CAT fusion genes with either 524 or 2300 base pairs of 5'-flanking DNA. In the presence of insulin, glucocorticoid or PRL addition alone increased endogenous beta-casein gene expression 2- to 2.5-fold and 5- to 10-fold, respectively, but only a 1.2- to 2.5-fold induction of CAT activity was observed for each hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.     

Identification of cis-acting DNA elements involved in the regulation of angiotensinogen gene expression.

Angiotensinogen is the precursor molecule of one of the most potent vasoactive substances, angiotensin-II. Angiotensinogen is normally synthesized in the liver and secreted into the plasma where it is converted into angiotensin-II by the combined proteolytic action of renin and angiotensin converting enzyme. Angiotensinogen levels in the plasma are modulated by a number of pathological and physiological factors. In order to understand the regulation of angiotensinogen gene expression, we have constructed an expression vector in which 688 bp of the 5'-flanking region of the rat angiotensinogen gene were attached to the chloramphenicol acetyl transferase (CAT) coding sequence. We have also obtained 5'-sequential deletion mutants from the rat angiotensinogen promoter attached to the CAT gene, and have identified multiple cis-acting DNA sequences involved in the regulation of angiotensinogen gene expression by transient transfection of these recombinant DNA molecules in human hepatoma cell lines, Hep3B, and HepG2.     

Initiation of translation by non-AUG codons in human T-cell lymphotropic virus type I mRNA encoding both Rex and Tax regulatory proteins

Human T-cell lymphotropic virus type I (HTLV-I) double-spliced mRNA exhibits two GUG and two CUG codons upstream to, and in frame with, the sequences encoding Rex and Tax regulatory proteins, respectively. To verify whether these GUG and CUG codons could be used as additional initiation codons of translation, two chimeric constructs were built for directing the synthesis of either Rex–CAT or Tax–CAT fusion proteins. In both cases, the CAT reporter sequence was inserted after the Tax AUG codon and in frame with either the Rex or Tax AUG codon. Under transient expression of these constructs, other proteins of higher molecular mass were synthesized in addition to the expected Rex–CAT and Tax–CAT proteins. The potential non-AUG initiation codons were exchanged for either an AUG codon or a non-initiation codon. This allowed us to demonstrate that the two GUG codons in frame with the Rex coding sequence, and only the second CUG in frame with the Tax coding sequence, were used as additional initiation codons. In HTLV-I infected cells, two Rex and one Tax additional proteins were detected that exhibited molecular mass compatible with the use of the two GUG and the second CUG as additional initiation codons of translation. Comparison of the HTLV-I proviral DNA sequence with that of other HTLV-related retroviruses revealed a striking conservation of the three non-AUG initiation codons, strongly suggesting their use for the synthesis of additional Rex and Tax proteins.     

Thyroid hormone-responsive elements of the prolactin gene: evidence for both positive and negative regulation

To analyze the regulation of PRL gene expression by thyroid hormone (T3), fusion gene constructs containing various lengths of the rat PRL gene 5'-flanking sequence linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were transfected into the GH3 cell line. Thyroid hormone had no effect on basal or cAMP-stimulated CAT expression in constructs containing more than 1.7 kilobasepairs of the 5'-sequence. However, deletion to 1.5 or 0.6 kilobasepairs resulted in an inhibition of both basal and cAMP-stimulated expression by T3. A construct containing the proximal enhancer region (positions -292 to -38 basepairs) linked to the herpes simplex thymidine kinase promoter (TK) and the CAT reporter gene also responded to T3 with inhibition of basal and cAMP-induced CAT expression. The distal enhancer region (positions -1714 to -1495) linked to thymidine kinase promoter CAT responded to T3 with a stimulation of CAT expression, and the response was additive with the stimulatory response to cAMP. Deletion analysis of the distal enhancer region revealed that the sequence between positions -1530 and -1565 was required for the stimulatory response to T3. The stimulatory response to T3 was additive with the response to estradiol, suggesting distinct elements, but deletion to position -1565 abolished the response to estradiol and permitted an inhibitory response to T3. Mutation of the estrogen response element prevents the response to estradiol, but only blunted the response to T3. Mutation of the sequence GGTCA at positions -1555 to -1551 resulted in an inhibitory response to T3, implicating this sequence in the stimulatory response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of microinjected hsp 70/CAT and hsp 30/CAT chimeric genes in developing Xenopus laevis embryos

The expression of microinjected chimeric genes containing Drosophila hsp 70 and Xenopus hsp 70 and hsp 30 promoters linked to the reporter gene coding for bacterial chloramphenicol acetyltransferase (CAT) was examined during early development of Xenopus laevis. Heat-inducible expression of fusion genes containing either the Drosophila hsp 70 promoter (1100 bp) or the Xenopus hsp 70 promoter (750 bp) was first detectable after the midblastula stage of development. This coincides with the embryonic stage at which the endogenous hsp 70 gene is first heat-inducible. A Xenopus hsp 30/CAT fusion gene containing 350 bp of promoter sequences was also heat-inducible after the midblastula stage unlike the endogenous hsp 30 genes which were not heat-inducible until the early tailbud stage (stage 23-24). Sequences that are present within either the coding or 3' region of the hsp 30 clone do not cause the microinjected hsp 30 gene to be developmentally regulated in a normal manner. Additionally, microinjected hsp 30 gene sequences have no effect on the developmental regulation of endogenous hsp 30 genes which continue to be activated at the tailbud stage of development. Our data suggest, that an inhibitory system, which may control the expression of the endogenous hsp 30 gene during development, does not regulate the expression of the injected hsp 30 gene.