Characterization of two cis-acting DNA elements involved in the androgen regulation of the probasin gene.

The location and sequence of androgen responsive elements (AREs) in the 5'-flanking DNA of the androgen-regulated rat probasin (PB) gene were determined. The DNA- and steroid-binding domains of the rat androgen receptor [glutathione-S-transferase (GST)-AR1] and the DNA-binding domain and hinge region alone (GST-AR2) were expressed in Escherichia coli as isopropyl-B-D-thioglactopyranoside-induced fusion proteins with GST and purified using glutathione affinity chromatography. Band shift assays indicated that the AR1 peptide was at least five times more effective than AR2 in binding to PB 5'-flanking DNA (-426 to +28), although both gave qualitatively similar patterns and were displaced by anti-AR antibodies. DNase I footprinting experiments revealed two putative AREs: one between positions -236 and -223 (ARE-1) and the other between -140 and -117 (ARE-2). Hormonal regulation of PB was determined by cotransfecting reporter constructions containing the PB 5'-flanking region (-426 to +28) linked to the bacterial chloramphenicol acetyl transferase (CAT) gene with androgen, glucocorticoid, or progesterone receptor expression vectors into human prostatic carcinoma cells (PC-3). PB-CAT gene expression was more effectively induced by androgens than by glucocorticoids or progestins. Both 5'- and 3'-deletion mapping of the PB 5'-flanking DNA revealed that ARE-1 and ARE-2 were required for androgen regulation. A single base mutation in either ARE resulted in a more than 95% loss of androgen induction of CAT. In comparable transfection experiments, the PB hormone-responsive elements showed a greater induction by androgens than did mouse mammary tumor virus or tyrosine aminotransferase elements. Thus, the preferential androgen regulation of the PB gene involves the participation of two different cis-acting DNA elements that bind AR.

Detection of placental growth hormone variant and chorionic somatomammotropin ribonucleic acid expression in human trophoblastic neoplasms by reverse transcriptase-polymerase chain reaction.

Attempts to assess human placental GH variant (hGH-V) and chorionic somatomammotropin (hCS) RNA in choriocarcinoma cell lines have been hampered by low levels of expression and limited sensitivity of RNA blotting analysis. We examined human choriocarcinoma BeWo, JAR, and JEG-3 cell lines as well as samples of complete hydatidiform moles for expression of members of the human GH (hGH) gene family using reverse transcriptase-polymerase chain reaction. A single and common set of primers was designed and used to detect products of the hGH/hCS genes as well as distinguish processed RNA from any contaminating DNA. Transcripts from the hCS genes hCS-A and -B were distinguished from placental hGH variant (hGH-V) and hCS-like (hCS-L) gene RNA by diagnostic restriction digestion of the polymerase chain reaction products. The expected pattern of hGH/hCS RNA expression was detected in term placenta, where hCS and hGH-V/hCS-L transcripts represented approximately 95% and approximately 5% of the total hGH/hCS RNA, respectively. The level of hCS RNA varied from 22-99% of the total hGH/hCS RNA in the neoplastic trophoblast samples, and variable levels of hGH-V and hCS-L RNA were also observed. In choriocarcinoma JAR cells, hGH-V RNA represented approximately 78% of the total hGH/hCS RNA compared to approximately 22% for hCS. Further, although low hCS-L RNA levels (< 1%) were found in term placenta and two of the hydatidiform moles, hCS-L transcripts represented 11% of the total hGH/hCS RNA in a third hydatidiform mole. Finally, in contrast to the detection of variable levels of hCS-L RNA in term placenta and hydatidiform mole samples, no hCS-L transcripts were detected in the three choriocarcinoma cell lines examined. These patterns reflect either deregulated hGH/hCS gene expression in neoplastic trophoblasts or differences that accompany the process of differentiation of trophoblast subpopulations. Regardless, this suggests that the control of hGH-V and hCS-L gene expression is distinct from that of the hCS-A and hCS-B genes and raises questions about the possible involvement of hGH/hCS family members in the pathology of placental abnormalities.

Efficient detection of unusual words.

Words that are, by some measure, over- or underrepresented in the context of larger sequences have been variously implicated in biological functions and mechanisms. In most approaches to such anomaly detections, the words (up to a certain length) are enumerated more or less exhaustively and are individually checked in terms of observed and expected frequencies, variances, and scores of discrepancy and significance thereof. Here we take the global approach of annotating the suffix tree of a sequence with some such values and scores, having in mind to use it as a collective detector of all unexpected behaviors, or perhaps just as a preliminary filter for words suspicious enough to undergo a more accurate scrutiny. We consider in depth the simple probabilistic model in which sequences are produced by a random source emitting symbols from a known alphabet independently and according to a given distribution. Our main result consists of showing that, within this model, full tree annotations can be carried out in a time-and-space optimal fashion for the mean, variance and some of the adopted measures of significance. This result is achieved by an ad hoc embedding in statistical expressions of the combinatorial structure of the periods of a string. Specifically, we show that the expected value and variance of all substrings in a given sequence of n symbols can be computed and stored in (optimal) O(n2) overall worst-case, O (n log n) expected time and space. The O (n2) time bound constitutes an improvement by a linear factor over direct methods. Moreover, we show that under several accepted measures of deviation from expected frequency, the candidates over- or underrepresented words are restricted to the O(n) words that end at internal nodes of a compact suffix tree, as opposed to the theta(n2) possible substrings. This surprising fact is a consequence of properties in the form that if a word that ends in the middle of an arc is, say, overrepresented, then its extension to the nearest node of the tree is even more so. Based on this, we design global detectors of favored and unfavored words for our probabilistic framework in overall linear time and space, discuss related software implementations and display the results of preliminary experiments.

A possible role for D8/PSF-A-like sequences in lactotroph versus somatotroph expression of the human prolactin gene.

The transcription factor GHF-1/Pit-1 is essential for the expression of GH and prolactin (PRL) by somatotrophs and lactotrophs respectively. However, PRL is not expressed in mature somatotrophs despite the presence of GHF-1/Pit-1. A possible mechanism is the presence of a somatotroph-specific repressor in the 5'-flanking sequences of the PRL gene. The region -3500/-1750 of the human (h) PRL gene is associated with negative regulatory activity and contains an element, designated D8, that resembles repressor PSF-A sequences which are located in the distal upstream region of placental members of the human GH family. An internal deletion of D8 sequences resulted in a significant stimulation of promoter activity in somatotroph GC (P < 0.005) and somatolactotroph-like GH3 and GH4C1 cells (P < 0.05), but not lactotroph-like 235-1 cells after gene transfer. However, D8 binding was observed by nuclease protection with lactotroph- as well as somatotroph-like cell nuclear protein. Although proteins that bind to the D8 element appear ubiquitous, this element does yield tissue-specific complexes in mobility shift assays. Further, competition studies do not suggest an interaction between GHF-1/Pit-1 and D8 proteins. The hPRL D8 element was inserted upstream of a thymidine kinase promoter and used to transfect pituitary and non-pituitary HeLa cells, to assess intrinsic repressor activity and/or promoter specificity. Although no repression was observed, a significant ninefold increase in expression was observed in HeLa cells (P < 0.001) which was at least twofold greater than observed in any of the pituitary cell lines tested. These results implicate D8 in the somatotroph-specific repression of hPRL; however, they also suggest that D8 can act as a stimulator as well as a repressor, depending on the interaction of a ubiquitous D8 factor forming promoter and cell-specific complexes with other elements/factors.

Chorionic gonadotrophin and c-myc expression in growing and growth-inhibited (intermediate) trophoblasts.

FEG-3 cells are a clonal line of human choriocarcinoma and resemble villous cytotrophoblasts which are the stem cells for the syncytiotrophoblast in the placenta. FEG-3 cells synthesize and secrete the alpha subunit of human chorionic gonadotrophin (hCG). Treatment of FEG-3 cells with the chemotherapeutic drug (1 microM) methotrexate (MTX) results in an increase in nuclear diameter. Cell division is blocked and a decrease in c-myc mRNA levels in observed. The effects on cell growth and c-myc mRNA expression are reversible, and cells treated with MTX for 48 h retain their proliferative potential. Assessment of placental hormone gene expression reveals that a member of the human growth hormone gene family is expressed at extremely low levels and is unaffected by MTX treatment. Alpha and beta chorionic gonadotrophin (hCG) levels are increased by MTX treatment, but levels decrease following removal of MTX. In contrast to hCG in FEG-3 cells, non-trophoblastic or ectopic production of alpha hCG in human cervical carcinoma cells is inhibited by MTX treatment. These data indicate that MTX will induce morphological and biochemical changes in FEG-3 cells. They reveal an inverse relationship between c-myc and hCG RNA expression, and suggest different mechanisms govern trophoblast versus non-trophoblast production of alpha hCG.

Differential expression of human placental growth hormone variant and chorionic somatomammotropin genes in choriocarcinoma cells treated with methotrexate.

Chorionic somatomammotropin (hCS) genes (hCS-A and hCS-B) and the placental growth hormone variant (hGH-V) gene are expressed in the syncytiotrophoblast in vivo, and at low levels in cytotrophoblast-like choriocarcinoma (BeWo) cells. Treatment of choriocarcinoma cells with methotrexate (MTX) will induce a cell type intermediate between a cytotrophoblast and syncytiotrophoblast. After treatment with MTX, hCS/hGH-V mRNA levels were decreased in BeWo cells, and only hGH-V and minor hCS-A related transcripts of 1.6, 2.1 and 4.2 kilobases, termed hCS-A2, hCS-A3 and hCS-A4, respectively, were detected. By contrast, chorionic gonadotropin RNA levels were increased. This pattern of hCS/hGH-V expression resembles that observed when BeWo cells are grown in thyroid hormone (T3)-depleted serum, where hGH-V/hCS RNA increases in response to T3. This increase is blunted by MTX treatment, but is not due to a decrease in number or affinity of T3 receptors. These data indicate that the hGH-V and hCS genes can be differentially regulated by MTX, and are consistent with MTX interfering with T3 responsiveness of these genes. Also, if BeWo cells treated with MTX do represent a transitional state, these data raise the possibility that hGH-V and hCS possess a different temporal pattern of expression in the developing trophoblast.

Detection of placental growth hormone variant and chorionic somatomammotropin-L RNA expression in normal and diabetic pregnancy by reverse transcriptase-polymerase chain reaction.

Diabetes is a common complication encountered during pregnancy. Earlier studies indicated that diabetic placentas bear morphological alterations consistent with modified placental differentiation, including alterations in the villous cellular content, structure, and total surface. Limited data associating the diabetic status with the expression of terminal placental differentiation markers are available. The human growth hormone/chorionic somatomammotropin (hGH/CS) family consists of five genes, one of which (GH-N) is expressed efficiently in pituitary while the other four (CS-A, B, L, and hGH-V) are expressed in placenta and represent ultimate placental differentiation markers. We developed and applied a sensitive RT-PCR method coupled with diagnostic restriction digestion to determine the relative levels of the hGH/CS family in normal pregnancies and examine whether their mRNA expression pattern is altered in pregnancies complicated by diabetes. We show that relative hCS-L content changes during placental development. Specifically, normal term placentas express higher relative levels of hCS-L, lower relative hGH-V levels and a 70-fold lower hGH-V/CS-L mRNA ratio compared to early placentas. Also, many term placentas from diabetic pregnancies express lower relative levels of hCS-L mRNA and a much higher hGH-V/CS-L mRNA ratio compared to normal term placenta, resembling more an early placenta pattern of expression. Thus, our study suggests that the expression of terminal placental differentiation markers, such as the hGH/CS genes, is altered in term placentas from these diabetics reflecting either impaired placental differentiation or post-differentiation impairment of normal placental function.

Rat prolactin-like protein A partial gene and promoter structure: promoter activity in placental and pituitary cells.

Rat prolactin-like protein A (rPLP-A) is a member of a rapidly expanding family of prolactin-related proteins that are expressed during pregnancy by the rat placenta according to specific developmental patterns. Although the factors involved in the pituitary-specific expression of the prolactin and growth hormone genes themselves have been extensively studied, essentially nothing is known of the factors responsible for the placental expression of these new family members. In this paper we describe the isolation of rPLP-A genomic clones, analyze a portion of the 5' flanking sequence of this gene and use the recently described rat choriocarcinoma cell line, Rcho, in transient transfection studies to show that a 975 base-pair (bp) fragment of 5' flanking sequence is sufficient to specify placental expression of the rPLP-A gene.

Characterization of fibroblast growth factor receptor 1 RNA expression in the embryonic mouse heart.

We used reverse transcriptase-polymerase chain reaction (RT-PCR) to clone fibroblast growth factor receptor (FGFR) 1 isoforms from embryonic mouse heart and as a more sensitive method to characterize FGFR1 RNA expression in embryonic and adult mouse hearts. We describe the cloning of both full-length short (2259 base pairs) and long (2526 base pairs) FGFR1 isoform cDNAs which generated 86 and 102 kilodalton proteins, respectively, following in vitro translation. An assessment of FGFR1 RNA indicates that FGFR1-IIIc is the major form in both the embryonic and adult heart but there is an approximately 8.5-fold decrease in RNA levels in the adult. Differential RNA blotting as well as RT-PCR analyses are consistent with a switch in the relative expression of the short versus long FGFR1 isoforms during heart development. The long isoforms are more abundant in the embryo and the short isoforms predominate in the adult. This may be important in the regulation of growth and development of the heart.

Differential binding of rat pituitary-specific nuclear factors to the 5'-flanking region of pituitary and placental members of the human growth hormone gene family.

Placental chorionic somatomammotropin (hCS-A or B) and growth hormone variant (hGH-V) are members of the human growth hormone family, and are related by structure and function to pituitary growth hormone (hGH-N). However, while the hGH-N gene is expressed specifically in the anterior pituitary, hGH-V and hCS are produced in the placenta. Hybrid hGH-N, hGH-V and hCS-A genes containing 5'-flanking sequences, including the endogenous promoter, are preferentially expressed in rat pituitary tumor (GC) cells, after gene transfer. Since interaction with a pituitary-specific protein (Pit 1) is required for efficient hGH-N as well as rat growth hormone (rGH) gene expression in GC cells, binding of pituitary proteins to the hGH-V and hCS-A promoter sequences was investigated. Rat Pit 1 binds at two locations on the hGH-N gene, a distal (-140/-107) and proximal site (-97/-66), in a similar manner to that observed with the rGH gene. By contrast, efficient Pit 1 binding was seen only to the distal site of the hGH-V gene and the proximal site of the hCS-A gene. Although binding of a protein to the distal hCS-A sequences was observed, the site of interaction was truncated (-140/-116), not pituitary-specific, and was more consistent with the binding of Sp1. These data indicate that rat Pit 1 binds to the placental hGH-V and hCS-A genes and correlates with their promoter activity in GC cells after gene transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of fibroblast growth factor receptor-1 isoforms in the mouse heart: evidence for isoform switching during heart development.

Basic (b) fibroblast growth factor (FGF) mediates various biological responses including mitogenesis and angiogenesis by binding to specific cell surface receptors of the tyrosine kinase family. The bFGF receptor-1 FGFR1) exists in short and long isoforms due to alternate RNA splicing. Minor alterations in the amino acid sequence have also led to reports of different FGFR1 isoforms in different tissues even in the same species. In the absence of any sequence for heart FGFR1 and accumulating evidence for a role of bFGF in heart growth and differentiation, we cloned FGFR1 from embryonic mouse hearts. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to generate full-length short (2259 base pairs) and long (2526 base pairs) forms of FGFR1 cDNAs which generated 86 and 102 kDa proteins, respectively, following in vitro translation. Embryonic mouse heart FGFR1 differed by seven amino acids from the reported sequence for mouse neuroepithelial FGFR1 and appeared more similar to human placental FGFR1. A single FGFR1 transcript of approximately 4.3 kb was seen in RNA isolated from embryonic as well as adult mouse hearts. There was a decrease (approximately 8.5-fold) in FGFR1 RNA levels in the adult. The majority of FGFR1 transcripts in the adult as well as embryonic heart contained exon IIIc (FGFR1-IIIc) which is associated with isoforms that display the highest affinity for bFGF. However, the relative ratio of short versus long FGFR1 RNA expression was 0.5 in the embryonic heart compared to 5.9 in the adult heart. These results indicate that: (i) structurally distinct short and long FGFR1 isoform RNAs are expressed in the embryonic and adult heart; (ii) FGFR1-IIIc is the major form of receptor expressed in the embryonic as well as adult heart; (iii) the transition from the embryo to the adult stage is associated with a decrease but not absence of FGFR1 RNA expression; and (iv) long FGFR1-isoforms are more abundant in the embryo while short FGFR1 isoforms predominate in the adult.

Effect of "enhancer" sequences on ventricular myosin light chain-2 promoter activity in heart muscle and nonmuscle cells.

Positive (HF-1 and HF-2) and negative (HF-3) elements responsible for cardiac-specificity of the rat ventricular myosin light chain-2 (MLC-2v) promoter are contained in a 250 base pair region. The effect of the simian virus 40 (SV40) enhancer or 3 copies of the HF-1, HF-2 and HF-3 elements on MLC-2v promoter activity and specificity was assessed in neonatal rat cardiac myocytes and cardiac nonmuscle cells as well as rat heart myoblast H9c2 and glioma (nonmuscle) C6 cell lines. The SV40 enhancer increased promoter activity by at least 10-fold in both muscle and nonmuscle cell types; however, there was a decrease in cardiac ventricular myocyte-specificity. In contrast, the 3 copies of HF-1, HF-2 and HF-3 elements stimulated MLC-2v promoter activity approximately 3-fold in neonatal ventricular cardiac myocytes alone and, effectively, displayed about a 5-fold increase in specificity over the wild type MLC-2v promoter.