The immediate-early gene Egr-1 regulates the activity of the thymidine kinase promoter at the G0-to-G1 transition of the cell cycle.

Production of thymidine kinase (TK) protein parallels the onset of DNA synthesis during the cell cycle. This process is regulated at transcriptional, posttranscriptional, and translational levels to cause a 40- to 50-fold increase in cytosolic enzymatic activity as cells progress from G1 to S phase. Transcriptional activation of the mouse TK gene through the cell cycle is dependent upon previously characterized cis elements of the proximal promoter, called MT1, MT2, and MT3, that bind at least two different complexes: TKE during the transition of cells from quiescence (G0) to G1, and Yi later at the G1/S boundary. To identify the transcription factors involved in this regulation, we screened a mouse fibroblast cDNA expression library with a labeled MT3 oligonucleotide probe and isolated a clone that encodes Egr-1, an immediate-early transcription factor, whose expression is regulated by serum or growth factors during the G0-to-G1 transition when cells reenter the cell cycle. Electrophoretic mobility shift assays demonstrate that Egr-1 is involved in the TKE complex that binds to the MT3 element and that expression of Egr-1 induces transcription of a mouse TK-chloramphenicol acetyltransferase reporter in transient transfections. These results suggest a role for Egr-1 in regulating expression of the TK gene at the G0-to-G1 transition.

Hormonal regulation of rat androgen-binding protein (ABP) messenger ribonucleic acid and homology of human testosterone-estradiol-binding globulin and ABP complementary deoxyribonucleic acids.

Complementary DNA clones coding for rat androgen-binding protein (rABP) were isolated from a rat testis cDNA library constructed in the bacteriophage lambda gt11. The library was screened immunochemically, using two different antibodies against rABP. The identity of the isolated clones was confirmed by epitope selection and DNA sequence analysis. The mRNA encoding rABP could be detected in the testes of 20- and 46-day-old-rats, but not in the 10-day-old rats by hybridization with 32P-labeled rABP cDNA in a Northern blot of poly(A)+-RNA fractioned by agarose gel electrophoresis. No hybridization signal was seen with poly(A)+-RNA isolated from kidney and liver. The rABP mRNA appeared as a single species with a size of 1.65 kilobase, sufficient to encode a protein of 42,000 daltons. The concentration of rABP mRNA in the testes of 37-day-old hypophysectomized rats increased after treatment with testosterone and FSH, given alone or in combination. Sequence and hybridization analysis of cDNAs for rABP, human testosterone-estradiol-binding globulin, and human ABP demonstrates that the cDNAs for human testosterone-estradiol binding globulin and human ABP have greater sequence similarity with each other than either has with rABP.

Processing of angiotensin II (A-II) and (Sar1,Ala8)A-II by cultured bovine adrenocortical cells.

Bovine adrenocortical cells, cultured in a chemically defined medium, were used to study the fate of [125I] iodoangiotensin II ([125I]iodo-A-II) and its antagonist (Sar1,Ala8)A-II ([125I]iodo-Saralasin). The binding of both ligands was time and temperature dependent. The maximum specific binding at 37 C, which was reached within 1 h, was followed by a decline with a half-life of about 2 h and 8 h for [125I]iodo-A-II and [125I]iodo-Saralasin, respectively. The decrease of the specific binding was parallel to the appearance in the medium of degraded ligand. At 4 C, the binding of [125I]iodo-A-II was stable for 12 h and no degradation of ligand occurred. Under several experimental conditions, about 70% of the total [125I]iodo-A-II bound was internalized, whereas, in the case of [125I]iodo-Saralasin, less than 25% of the total bound ligand was internalized. These differences in the binding kinetics between A-II and its antagonist were mainly the differences in the rate of internalization of the bound ligands, more rapid for [125I]iodo-A-II (t1/2 approximately equal to 10 min) than for [125I]iodo-Saralasin (t1/2 = 90 min). On the other hand, the rate of degradation of internalized ligand was similar for both ligands (t1/2 = 15 min). Ionophore monensin enhanced the total cellular uptake of both ligands by increasing the amount of internalized ligands. Monensin did not modify the rate of internalization of the two ligands but markedly decreased their rate of degradation (t1/2 approximately equal to 60 min). These results indicate that both A-II and its antagonist are internalized and degraded by adrenocortical cells, but the rate of internalization of the antagonist is lower than that of the agonist. They also show that receptor-mediated endocytosis is the main pathway by which A-II is rapidly degraded by adrenocortical cells. Since A-II receptors are present in many tissues, the receptor-mediated degradation could explain the very short half-life in plasma of this hormone.

In vitro spontaneous and adrenocorticotropin-dependent maturation of the steroidogenic pathway of ovine fetal adrenal cells.

Adrenal cells from intact or hypophysectomized fetuses and newborn lambs cultured for 6 days under several conditions were tested for their ability to metabolize [14C]pregnenolone (P5) (5 X 10(-5) M) during a 2-h period. Also, their ability to produce P5 was investigated. At the beginning of the culture, the metabolism of P5 by adrenal cells from hypophysectomized fetuses was lower than that of cells from intact fetuses which in turn was lower than that of cells from newborns. After 6 days in culture in the absence of ACTH-(1-24), the metabolism of P5 by cells from both intact and hypophysectomized fetuses increase, whereas that of cells from newborns slightly decreases. Continuous ACTH-(1-24) (10(-8) M) treatment produced a striking increase in the capacity to metabolize P5 by cells from both intact and hypophysectomized fetuses and the maintainance of this capacity of cells from newborns was demonstrated. At the beginning of the culture the main product of [14C]P5 was corticosterone with cells from intact and hypophysectomized fetuses, and cortisol with newborn cells. After 6 days in culture in ACTH-free medium more than 50% of P5 added appeared as progesterone in all cases. Addition of ACTH-(1-24) to the culture medium induced within 5 days, a striking increase in the activation of 3 beta-hydroxysteroid dehydrogenase-isomerase, 17 alpha-, 21, and 11 beta-hydroxylases of adrenal cells from fetuses. However, the activity of 17 alpha-hydroxylase in adrenal cells from hypophysectomized fetuses was lower than that of cells from intact fetuses. ACTH-(1-24) treatment maintained the enzymatic activity of cultured newborn adrenal cells, except for 11 beta-hydroxylase which declined to about 50% of its activity on day 0. The capacity of fetal adrenal cells to produce P5 after acute ACTH-(1-24) stimulation increased 30-fold when cells were cultured in the absence of ACTH and up to 110-fold when cells were treated with ACTH-(1-24) from day 1 on. These results, together with those reported previously, show that the spontaneous in vitro maturation of fetal adrenal cell steroidogenic pathway is related to an increased activity of the 3 beta-hydroxylase dehydrogenase-isomerase and an enhanced capacity to produce P5. Both steps were stimulated further by ACTH. In addition, the hormone is an absolute requirement for the development and/or the maintenance of 17 alpha-hydroxylase activity.

Comparison of androgen regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase gene expression in rodent kidney and accessory sex organs.

Androgen regulation of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities and accumulation of the mRNAs encoding these two enzymes in rodent kidney and accessory sex organs were studied. The ODC mRNA concentration and enzyme activity were increased by androgens in kidney, prostate, and seminal vesicle of 3-day castrated mice and rats, whereas AdoMetDC mRNA and protein levels were androgen inducible only in rodent accessory sex organs. ODC and AdoMetDC mRNAs were regulated in prostate and seminal vesicles in a coordinate fashion, with the maximal levels reached within 24-48 h of steroid exposure. The extent of induction was similar for the two gene products, and ODC and AdoMetDC mRNA accumulation occurred primarily in epithelial cells of accessory sex organs, as shown by in situ hybridization studies. The murine ODC promoter contains an androgen-responsive element (ARE)-like sequence at about -910 nucleotides from the cap site; this element binds to androgen receptor in vitro, albeit with much lower affinity than some other AREs. In transient expression studies with CV-1 cells, an ODC promoter construct (pODCCAT) conferred androgen responsiveness upon the reporter gene. ODC mRNA accumulation is androgen regulated in epithelial cells of proximal tubules in both murine and rat kidney; however, different subtypes of the proximal tubular cells respond in the two species, as revealed by in situ hybridization studies. Expression of the ODC gene was induced more markedly and for a longer duration in the murine than in the rat kidney, but the initial response occurred faster in the rat kidney. The relatively slow kinetics of ODC mRNA accumulation in mouse kidney were not due to recruitment of new cells to respond; rather, in situ hybridization studies indicated that there was progressive accumulation of the mRNA in the responding cells. Collectively, these data indicate that the genes for two key enzymes in polyamine biosynthesis are not regulated in an identical fashion in different androgen target tissues, such as rodent kidney and accessory sex organs.

Estrogen regulates the stage-specific expression of kidney androgen-regulated protein in rat uterus during reproductive cycle and pregnancy.

The female sex steroid, estrogen, acting through its nuclear receptor profoundly influences growth and differentiation programs in the mammalian uterus by regulating the expression of specific cellular genes. The identity and profile of expression of the estrogen-regulated genes at various stages of the reproductive cycle and pregnancy, however, remain largely unknown. Using a differential gene expression screen method, we isolated a gene that is down-regulated in the uterus during pregnancy. This gene encodes the previously identified androgen-regulated protein known as the kidney androgen-regulated protein (KAP) because of its abundant expression in the kidney. Our results showed a high level of KAP messenger RNA (mRNA) in the uteri of nonpregnant rats at all stages of the estrous cycle. We observed that in the pregnant animals, the level of KAP mRNA remained high immediately after fertilization (days 1 and 2), but declined sharply with the progression of pregnancy, falling to almost undetectable levels during midgestation (days 10-15). Interestingly, the level of KAP mRNA started to rise again toward the end (day 19 onward) of pregnancy and was high during parturition. The temporal pattern of expression of KAP in the uterus closely overlapped with the profile of plasma estrogen during pregnancy. Known antagonists of estrogen, such as tamoxifen and ICI 182,780, strongly inhibited uterine KAP gene expression during the estrous cycle and pregnancy, supporting a regulatory role of estrogen in this process. Consistent with this observation, administration of estrogen to ovariectomized animals markedly stimulated (approximately 10-fold) the level of KAP mRNA in the uterus. Treatment of these animals with progesterone, on the other hand, did not significantly after KAP gene expression. Immunocytochemical analyses of uterine sections with an antibody against KAP exhibited specific staining in both luminal and glandular epithelia and in myometrium. These uterine locations also possess abundant estrogen receptors and are known targets of estrogen action. Our studies, therefore, revealed that KAP is a useful marker of estrogen action in the uterus, especially during the reproductive cycle and termination of gestation.

Human ornithine decarboxylase-encoding loci: nucleotide sequence of the expressed gene and characterization of a pseudogene.

Previous studies have shown that human ornithine decarboxylase (ODC)-encoding sequences map to two chromosome regions: 2pter-p23 and 7cen-qter. In the present work we have cloned the expressed human ODC gene from a genomic library of myeloma cells that overproduce ODC protein due to selective gene amplification and determined its entire nucleotide sequence. The gene comprises 12 exons and 11 introns and spans about 8 kb of chromosome 2 DNA. The organization of the human gene is very similar to that of the mouse and rat, with the major difference being the presence of longer intronic sequences in the human gene. Some of these differences can be accounted for by the insertion of four Alu sequences in the human gene. Several potential regulatory elements are present in the promoter region and in 5'-proximal introns, including a TATA box; GC boses; AP-1-, AP-2- and NF-1-binding sites; and a cAMP-responsive element. The 5'-untranslated sequence of ODC mRNA is extremely GC-rich, and computer predictions suggest a very stable secondary structure for this region, with an overall free energy of formation of -225.4 kcal/mol. In addition to the active ODC gene on chromosome 2, ODC gene-related sequences were isolated from human chromosome 7-specific libraries and shown to represent a processed ODC pseudogene.

Identification by molecular cloning of two forms of the alpha-subunit of the human liver stimulatory (GS) regulatory component of adenylyl cyclase.

Two DNA molecules complementary to human liver mRNA coding for the alpha-subunit of the stimulatory regulatory component Gs of adenylyl cyclase were cloned. One of the two forms is a full-length cDNA of 1614 nucleotides plus a poly(A) tail of 59 nucleotides. The deduced sequence of 394 amino acids encoded by its open reading frame is essentially identical to that of the alpha-subunits of Gs identified by molecular cloning from bovine adrenals, bovine brain and rat brain. Two independent clones of the other type of cDNA were isolated. Both were incomplete, beginning within the open reading frame coding for the alpha s polypeptide. One codes for amino acids 5 through 394 and the other for amino acids 48 through 394 of the above described cDNA of 1614 nucleotides, and both have the identical 3'-untranslated sequence. They differ from the first cDNA, however, in that they lack a stretch of 42 nucleotides (numbers 214 through 255) and have nucleotides 213 (G) and 256 (G) replaced with C and A, respectively. This results in a predicted amino acid composition of another alpha-subunit of Gs that is shorter by 14 amino acids and contains two substitutions (Asp for Glu and Ser for Gly) at the interface between the deletion and the unchanged sequence. We call the smaller subunit alpha s1 and the larger alpha s2. This is the first demonstration of a structural heterogeneity in alpha s subunits that is due to a difference in amino acid sequence.

Androgen-regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase genes.

Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) are two key enzymes in polyamine biosynthesis. Both the ODC and the AdoMetDC gene is regulated by androgens in accessory sex organs of mice and rats, whereas only the ODC gene is androgen-responsive in rodent kidney. Androgenic responses in murine and rat kidneys are, however, dissimilar in that the induction of ODC activity and ODC mRNA accumulation is transient in the rat but sustained in the murine renal cells. In addition, in situ hybridization experiments with single-stranded cRNA probes revealed that ODC gene expression occurs in different subpopulations of epithelial cells of the proximal tubules in mice and rats. ODC and AdoMetDC genes are androgen-regulated in the same cell types of the accessory sex organs, as judged by hybridization histochemistry. Sequencing of the promotor region of the murine ODC gene has indicated the presence of several DNA elements for binding of transcription factors/regulatory proteins, including a putative androgen-response element at about 900 nucleotides upstream of the transcription start site.

7alpha-methyl-19-nortestosterone, a synthetic androgen with high potency: structure-activity comparisons with other androgens.

CNNT. There was a good correlation between bioactivity and binding affinity to AR for the 7alpha-substituted androgens compared to T. In contrast, relative to their binding affinity to AR, the androgenic potency of DHT and 19-NT was lower compared to T. The reason for the lower in vivo androgenic activity of 19-NT is attributable to its enzymatic conversion to 5alpha-reduced-19-NT in the prostate. In the case of DHT, the lower bioactivity could be attributed to its faster metabolic clearance rate relative to T. The correlation was further investigated in vitro by co-transfection of rat ARcDNA expression plasmid and a reporter plasmid encoding the chloramphenicol acetyl transferase (CAT) gene driven by an androgen inducible promoter into CV-1 cells. All the androgens led to a dose-dependent increase in the CAT activity. MENT was found to be the most potent followed by DHT, 19-NT, T, and CNNT. The specificity of the androgenic response was confirmed by its inhibition with hydroxyflutamide, an antiandrogen. Thus, there was a good correlation between binding affinity and in vitro bioactivity in the transient transfection assay for the androgens. This suggests that the in vivo bioactivity of androgens could be influenced not only by binding affinity to receptors but also by factors such as absorption, binding to serum proteins and metabolism. However, the high potency of MENT is primarily related to its higher affinity to AR.

Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma.

Human myxoid liposarcomas contain a characteristic chromosomal translocation, t(12;16)(q13;p11), that is associated with a structural rearrangement of the gene encoding CHOP, a growth arrest and DNA-damage inducible member of the C/EBP family of transcription factors residing on 12q13.1. Using a CHOP-specific complementary probe and antiserum we report here the presence of an abnormal CHOP transcript and protein in these tumours. Cloning of the translocation-associated CHOP gene product revealed a fusion between CHOP and a gene provisionally named TLS (translocated in liposarcoma). TLS is a novel nuclear RNA-binding protein with extensive sequence similarity to EWS, the product of a gene commonly translocated in Ewing's sarcoma. In TLS-CHOP the RNA-binding domain of TLS is replaced by the DNA-binding and leucine zipper dimerization domain of CHOP. Targeting of a conserved effector domain of RNA-binding proteins to DNA may play a role in tumour formation.

Cell-specific and hormonal regulation of the rat kidney androgen-regulated protein (KAP) gene.

In mouse kidney, the kidney androgen-regulated protein (KAP) gene is regulated in a sex-dependent manner by a complex tissue- and cell-specific multihormonal system. KAP is also found in mouse uterus during the period surrounding birth. We describe here for the first time the existence of KAP in a species other than mouse. The rat cDNA sequence was determined and the derived peptide sequence displayed only 53% identity with murine KAP, although the genomic organization of the genes was identical. Expression of rat KAP was restricted to kidney and uterus, but was constitutive in the latter and drastically induced at parturition. The renal expression of the rat KAP gene was sexually dimorphic and regulated by physiological levels of steroid hormones. The effects of castration, hypophysectomy, thyroidectomy, and castration plus thyroidectomy on KAP mRNA levels in both kidney and uterus were determined. Constitutive expression of the protein was strictly dependent on thyroid hormone in female kidneys where it was modulated by estrogens and other ovarian factor(s). In the uterus, KAP mRNA was mainly under estrogen control. In males, expression of the KAP gene was under the dual regulation of thyroid hormone and androgens. Its complex regulation suggests a carefully delineated role for KAP in the kidney and uterus, but its physiological function remains unknown.

Inhibitory or stimulatory effect of human genes on the expression of adrenal function in human Leydig X Y1 cell hybrids.

In order to investigate the expression and the regulation of steroidogenesis, human Leydig cells were fused with a functional mouse adrenal cell line (Y1). Six independent hybrid clones were analysed for hormone receptors and for cAMP and steroid response to ACTH, hCG, 8Br-cAMP or forskolin. All hybrids had lost hCG receptors and their ability to produce testosterone. With respect to the response of adenylate cyclase to ACTH and/or forskolin, hybrids could be classed into two groups. In the first group, the pattern of response was qualitatively similar to Y1 parental cells; The second group was far less responsive to ACTH than are Y1 cells, and when added together, forskolin and ACTH only had an additive effect. All hybrids responded to ACTH and 8Br-cAMP with an increased production of pregnenolone (P5). The amounts of P5 produced both under basal conditions and following 8Br-cAMP stimulation were significantly higher in three hybrids when compared to Y1 cells. However, the ability of two of these three hybrids to produce 20 alpha-dihydroprogesterone (20 alpha OHP4) was very low. The metabolism of [14C]P5 revealed that in one of these hybrids, there was a loss of 3 beta-hydroxysteroid dehydrogenase/isomerase whereas in the other case, there was a low 20 alpha-hydroxylase activity. The inhibition of cell growth by ACTH was related to the ability of the hormone to stimulate cAMP. Conversely, the inhibitory growth effects of 8Br-cAMP were not always inversely correlated with the ability of this nucleotide to stimulate P5 production. Since hybrids contained two mouse genomes and retained variable human chromosomes, these results suggest that extinction or enhancement of murine genes coding for some of the enzymes involved in steroidogenic response to ACTH was due to the regulation by human genes.

Regulation of angiotensin II receptors and steroidogenic responsiveness in cultured bovine fasciculata and glomerulosa adrenal cells.

The aim of the present study was to investigate the effect of several effectors on angiotensin II (A-II) receptors and steroidogenic responsiveness in cultured bovine fasciculata cells. Treatment of adrenal cells for 24 h with A-II (0.1 microM), corticotropin (1 nM), phorbol ester (PMA 0.1 microM), calcium ionophore A23187 (0.1 microM) and cyclic 8-bromoAMP (1 mM) produced a loss of A-II receptors whereas the A-II antagonist [Sar1-Ala8]A-II (0.1 microM) led to a small but significant increase. The extent of the down-regulation of receptors following maximal concentrations of A-II was greater than that produced by the other agents. The effects of A-II were dose-dependent with a ID50 of 3 nM. Since cycloheximide and actinomycin blocked the down-regulation of receptors, it seems likely that the effectors lead to the synthesis of certain proteins which inhibit the recycling of internalized receptors. Pretreatment of adrenal cells with A-II induced both homologous (90% decrease) and heterologous (corticotropin 83, PMA and ionophore 76% decrease) steroidogenic desensitization. However, the cAMP response to corticotropin of A-II-pretreated cells was higher (P less than 0.001) than for control cells. Pretreatment with PMA and A23187 also resulted in both homologous and heterologous steroidogenic refractoriness but to a lesser degree than that induced by A-II. In contrast, corticotropin-pretreated cells responded normally to further stimulation with corticotropin or A-II. Similarly pretreatment of bovine adrenal glomerulosa cells with A-II (1 nM and 0.1 microM) and corticotropin (1 nM) also induced A-II receptor loss and steroidogenic refractoriness. The present findings indicate that, in contrast to the results reported in vivo in the rat, where A-II leads to up-regulation of its own receptors on glomerulosa cells and increases steroidogenic responsiveness, this peptide results in both down-regulation and desensitization in cultured bovine fasciculata and glomerulosa cells. Our results also emphasize the absence of correlation between A-II receptor loss and steroidogenic responsiveness.

Structure and organization of the human S-adenosylmethionine decarboxylase gene.

Genomic clones for the S-adenosylmethionine (AdoMet) decarboxylase gene were isolated from a human chromosome 6 DNA library. In addition, polymerase chain reaction and specific primers were used to amplify fragments from chromosomal DNA covering exonic regions not found in the screening of DNA libraries with AdoMet decarboxylase cDNA. The gene encompasses at least 22 kilobases of chromosome 6 DNA and comprises nine exons and eight introns, in contrast to the corresponding rat gene that has only eight exons (Pulkka, A., Ihalainen, R., Aatsinki, J., and Pajunen, A. (1991) FEBS Lett. 291, 289-295). Exon-intron junctions in the human and rat AdoMet decarboxylase genes were in identical positions except that exons 6 and 7 of the human gene formed a single exon in the rat gene. Alu-like sequences are present in four introns and the 5'-flanking region of the human gene. The promoter region contains a TATA box adjacent to the cap site; in addition, DNA elements for binding of transcription factors AP-1, AP-2, CREB, SP-1, and multiple steroid receptors are present between position -3,158 and the transcription start site. Two AdoMet decarboxylase promoter-reporter gene constructs with about 170 and 1,500 nucleotides of the 5'-flanking DNA were used in transient expression studies. AdoMet decarboxylase promoter was capable of driving reporter gene expression, but it was less active than the murine ornithine decarboxylase promoter. There are at least three potential polyadenylation signals at the 3'-end of the gene, and utilization of the first two results in the formation of the 2.0- and 3.6-kilobase AdoMet decarboxylase mRNA species present in human tissues and cell lines. AdoMet decarboxylase gene-related sequences were also present in a human X chromosome-specific DNA library. Partial nucleotide sequencing of this DNA revealed a lack of introns present in the gene located on chromosome 6, suggesting that the locus on the X chromosome contains a processed AdoMet decarboxylase pseudogene.

CHOP (GADD153) and its oncogenic variant, TLS-CHOP, have opposing effects on the induction of G1/S arrest.

The growth arrest and DNA damage-inducible gene CHOP (GADD153) encodes a small nuclear protein from the C/EBP family, originally isolated from adipocytes in culture. Although inactive in cells under normal conditions, the CHOP gene is markedly induced by a variety of cellular stresses, including nutrient deprivation and metabolic perturbations. These lead to accumulation of CHOP protein in the nucleus. Because cellular stress normally leads to growth arrest, we examined the implication of CHOP in this process. Microinjection of CHOP expression plasmids into NIH-3T3 cells blocked the cells from progressing through the cell cycle, measured by an attenuation in the fraction of cells incorporating BrdU, an S-phase marker. The precise point in the cell cycle at which CHOP acts was mapped by microinjection of bacterially expressed CHOP protein into synchronized cells--this blocked the cells from progressing from G1 to S phase. This effect of CHOP was observed only when the protein was introduced early after serum stimulation suggesting that CHOP works at or around the so-called G1/S checkpoint. CHOP dimerizes with other C/EBP proteins and the CHOP-C/EBP dimers are directed away from "classical" C/EBP sites recognizing instead unique "nonclassical" sites. Mutant forms of the CHOP protein that lack the leucine zipper dimerization domain or the unusually structured basic region, potentially involved in DNA binding, fail to induce growth arrest. A tumor-specific form of CHOP, TLS-CHOP, that has been found so far exclusively in the human adipose tissue tumor myxoid liposarcoma, fails to cause growth arrest and furthermore interferes with the ability of normal CHOP to induce growth arrest. CHOP has been shown recently to be markedly inducible by nutritional deprivation of cells. This suggests that CHOP may play a role in an inducible growth arrest pathway that is triggered by metabolic cues and is of particular importance in adipose tissue--an organ that undergoes marked changes in its metabolic activity. Blocking of this pathway by TLS-CHOP may play a mechanistic role in the establishment of myxoid liposarcoma.

The human S-adenosylmethionine decarboxylase gene: nucleotide sequence of a pseudogene and chromosomal localization of the active gene (AMD1) and the pseudogene (AMD2).

S-adenosylmethionine decarboxylase (AdoMet-DC) is a key enzyme in polyamine biosynthesis. The human genome contains at least two loci for the AdoMetDC gene (AMD), one of which (AMD1) has previously been mapped to chromosome 6 and the other (AMD2) to the X chromosome. The locus on chromosome 6 is the transcriptionally active gene. We now report characterization of the AMD2 locus (GenBank Accession No. U02035) on the X chromosome, which contains sequences that cross-hybridize with human AdoMetDC cDNA. This DNA lacks all of the introns present in AMD1 and has numerous mutations in the protein-coding region. Its overall nucleotide sequence identity with AdoMetDC cDNA is about 90%. AMD2 is therefore a processed pseudogene, which, because of multiple mutations, cannot be translated to an active AdoMetDC enzyme, even if it were transcribed. Chromosomal loci for human AMD sequences were determined by in situ hybridization to metaphase chromosomes, with genomic DNAs from the active gene and the pseudogene loci as probes. AMD1 was localized to chromosome region 6q21-->q22 and AMD2 to band Xq28.

Association of the mammalian helicase MAH with the pre-mRNA splicing complex.

Conversion of pre-mRNAs into mature mRNAs includes several consecutive enzymatic modification steps that are carried out in the spliceosomes. Helicases have been shown to contribute to these catalytic processes both in yeast and in mammalian cells. Our results identify the mammalian protein MAH (matrix-associated helicase) as a new helicase present in the spliceosome complex. Sequence comparison describes MAH as the first higher eukaryotic member of the helicase superfamily I, with demonstrated enzymatic activity. Because MAH does not bind small nuclear ribonucleoproteins (snRNPs), it appears to be a non-snRNP binding factor of the splicing complex. In conclusion, our data suggest the involvement of MAH in processing of pre-mRNAs in mammalian cells.