Human Growth Hormone Gene Research Articles

Viability of transgenic rat embryos after freezing and thawing.

In-vivo viability of frozen-thawed embryos derived from transgenic rats, as well as the transmission and the expression of transgenes in the resultant newborn rats, was investigated. Three strains of transgenic rats, carrying human growth hormone gene connected downstream to the promoter region of the bovine alpha-lactalbumin gene (alpha LA/hGH), bovine beta-casein gene (beta CN/hGH) or bovine alpha-S1 casein gene (alpha S1CN/hGH), were used. Two-cell stage embryos (non-transgenic Wistar female x heterozygous transgenic male) were placed in 10% (v/v) dimethylsulfoxide (DMSO) solution and cooled from -7 to -30 degrees C at -0.5 degree C/min before being plunged into liquid nitrogen. After 2 to 4 years storage, the embryos were thawed by rapid warming. The intact embryos were transferred into the oviducts of Day 1 pseudopregnant recipients. The postthaw survival rate of frozen embryos was high in all 3 transgenic strains (88 to 92%), which was similar to that of control (non-transgenic) frozen embryos (95%). Development to newborn rats following transfer of embryos derived from the 3 strains (64 to 68%) was also similar to that of control embryos (60%). These transgenes (alpha LA/hGH, beta CN/hGH and alpha S1CN/hGH) were detected in the DNA extracts from tail tissue of the newborn rats, but the transmission rates (41, 23 and 32%, respectively) were lower than 50% which is expected in the Mendelian fashion. In a transgenic line carrying alpha S1CN/hGH, hGH levels of secretion into the milk of transgenic newborn rats derived from frozen-thawed embryos and her transgenic offspring were the same mg/ml-level as that of their founder rat. Two-step freezing of embryos derived from transgenic rats was therefore an effective method for the long-term cryopreservation of transgene.

Studies on the Production of Transgenic Mice

This paper describes attempts to identify parameters that are critical for succesful gene transfer into the germ line of mice by microinjection of foreign DNA into fertilized eggs. In the first stage of the study, 1470 zygotes were obtained and 1269 (86%) were microinjected with a human growth hormone gene construct. Of these zygotes, 769 (60%) proved to be alive after the injection, and 420 of them were transfered into the pseudopregnant recipients (54%). As a result, 70 offsprings were obtained, and out of these 3 (4%) proved to be transgenic. In the second stage of the study, 1246 zygotes were obtained and 1020 (81%) were microinjected with a human p53 gene construct. Of these zygotes 601 (58%) proved to be alive after the injection and 400 (66%) of them were transfered into pseudopregnant recipients. As a result, 105 (26%) offsprings were obtained, and out of these, 6 (5%) proved to be transgenic. Comparison of the ratios of transgenic mice in both groups showed no significant difference (P>0.05).

Ligase chain reaction assay for human mutations: the Sickle Cell by LCR assay

We can detect the beta-globin gene sickle cell mutation by using an assay based on the ligase chain reaction. The simultaneous amplification of the human growth hormone gene in the same reaction serves as a control for the amount of template DNA or amplification efficiency. Ligation products, which are biotinylated at one end and tagged with an arbitrary "tail" sequence at the other, are captured by hybridization to "tail"-complementary oligonucleotides immobilized on polystyrene microwells. The captured ligation products are detected colorimetrically by use of streptavidin-alkaline phosphatase conjugate. In a study of 24 subjects, the assay unequivocally discriminated among normal, carrier, and sickle cell genotypes.

Mechanisms responsible for dominant expression of human growth hormone gene mutations

Mechanisms responsible for dominant expression of human growth hormone gene mutations

Dopaminergic receptors and angiotensinogen gene expression in opossum kidney cells.

To investigate whether expression of the renal angiotensinogen gene is regulated by dopaminergic receptors, we used opossum kidney (OK 27) cells with a fusion gene containing the 5'- flanking regulatory sequence of the rat angiotensinogen gene fused with a human growth hormone (hGH) gene as a reporter [pOGH, angiotensinogen nucleotide (N) -1498/+18], permanently integrated into their genomes. The level of expression of pOGH (angiotensinogen N-1498/+18) in OK 27 was evaluated by the amount of immunoreactive hGH (ir-hGH) secreted into the culture medium. In the absence of 3-isobutyl-1-methylxanthine (IBMX), addition of dopamine (10(-13) to 10(-5)M) had minimal effect on the expression of the pOGH (angiotensinogen N-1498/+18) in OK 27 cells. In the presence of IBMX, addition of low concentrations (10(-13) and 10(-7) M) of dopamine stimulated the expression of pOGH angiotensinogen N-1498/+18) in OK 27 cells in a dose-dependent manner, whereas high concentrations (i.e., > 10(-7) M) had minimal effect. The stimulatory effect of dopamine on the expression of pOGH (angiotensinogen N-1498/+18) was inhibited by the presence of SCH-23390 (D1-dopaminergic receptor antagonist) and spiperone (D2-dopaminergic receptor antagonist), but not by ketanserin (5 HT2/5HT1c-serotonergic receptor antagonist). Moreover, the stimulatory effect of dopamine was inhibited by the presence of U-73122 (an inhibitor of phospholipase C and phospholipase A2) or staurosporine (an inhibitor of protein kinase C) or (R)-p-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMP[S]; an inhibitor of cAMP-dependent protein kinase AI and II). Addition of low concentrations (10(-13) to 10(-9)M) of SKF-82958 (D1-dopaminergic receptor agonist) or PPHT (D2-dopaminergic receptor agonist) also stimulated the expression of pOGH (angiotensinogen N-1498/+18). The stimulatory effect of SKF-82958 was inhibited by the presence of SCH-23390 or Rp-cAMP[S], whereas the effect of PPHT was inhibited by the presence of spiperone or staurosporine. These studies demonstrate that the expression of pOGH (angiotensinogen N-1498/+18) in OK 27 cells is modulated by dopaminergic receptor agonists.

Monitoring the transfection efficiency of the human follicle-stimulating hormone receptor cDNA in COS-7 cells: evaluation of the growth hormone transient gene expression assay system.

The human growth hormone (GH) transient gene expression assay system is frequently used to monitor transfection efficiency in transient transfection experiments. In this paper, we analyzed the suitability of the GH reporter gene to monitor transfection efficiency in COS-7 cells of an expression vector carrying the cDNA for the normal and mutated human follicle-stimulating hormone receptor (FSHR). The FSHR cDNA was cloned in the pSG5 expression vector and mutagenized (Ala307-->Thr) by oligonucleotide-mediated, site-directed mutagenesis. The expression plasmid pXGH5, carrying the structural gene for human GH, was used to monitor transfection efficiency. Different concentrations of pXGH5 and pSG5 containing normal or mutated FSHR cDNA were transfected in COS-7 cells by lipofection. The results showed: 1) The expression of pXGH5 was constant within individual experiments, but only in culture wells cotransfected with the same type of FSHR construct. On the contrary, the GH values normalized by the cell densities changed consistently depending on the type of FSHR construct. 2) The expression of the GH plasmid was influenced by type and concentration of the cotransfected plasmid. 3) The expression of pXGH5 cotransfected with the same FSHR construct was quite variable between experiments, without any relationship to the type of FSHR construct. These data show that the GH secretion is not a good parameter to monitor the transfection efficiency of the FSHR in pSG5 in COS-7 cells. Nor are other parameters such as semiquantitative mRNA determination or ligand binding to the transfected receptor ideal when mutations resulting in changes in receptor function are expected.

A modified quantitative polymerase chain reaction assay for measuring gene-specific repair of UV photoproducts in human cells.

Methods for measuring the induction and repair of ultraviolet (UV) induced modifications in short DNA fragments are essential for the study of gene-specific DNA repair. Measurements in genomic fragments of 14 kilobases (kb) or larger can be obtained using the enzyme-sensitive site (ESS) assay introduced by Hanawalt and Bohr (Bohr et al., 1985). More recently, several assays based on variations of the polymerase chain reaction (PCR) technique have been developed which have increased sensitivity (Govan et al., 1990; Kalinowski et al., 1992; Jennerwein and Eastman, 1991), even nucleotide resolution (Pfeifer et al., 1993). However, examination of these reports indicates that the PCR based DNA repair assays lack precision (Govan et al., 1990; Kalinowski et al., 1992; Tornaletti and Pfeifer, 1994; Jennerwein and Eastman, 1991). We report here, the development of a highly precise QPCR DNA repair assay. The assay was used to measure the induction and repair of UV photoproducts in a 2.7 kb genomic fragment containing the human growth hormone (hGH) gene in SL89 (wild-type) fibroblasts. The assay was exceedingly reproducible with an overall coefficient of variation from the mean of about 2.5%. This level of precision enabled the apparent simultaneous resolution of cyclobutane dimer (CPD) and (6-4) photoproduct (6-4PP) induction and repair.

Expression of Human Growth Hormone in Silkworm Larvae through RecombinantBombyx moriNuclear Polyhedrosis Virus

We have generated a recombinantBombyx morinuclear polyhedrosis virus, vBmhGH, harboring the full-length human growth hormone gene (2.4-kb genomic DNA, with four introns and the signal peptide sequences) under the control of the polyhedrin promoter. BmN cells in culture infected with the recombinant virus showed the presence of RNA corresponding to the authentic growth hormone mRNA as well as its incompletly processed precusor. Electrophoretic analysis and immunoprecipitation of proteins of recombinant virus-infected BmN cells revealed the presence of the growth hormone protein. Infection of silkworm larvae with vBmhGH led to the synthesis and efficient secretion of the protein into hemolymph. The recombinant human growth hormone was biologically active in a radioreceptor competition binding assay. The secreted protein was isolated and purified to homogeneity by a single step immunoaffinity chromatography, to a specific activity of 2.4 × 104U/mg. The recombinant hGH retained the immunological and biolological properties of the native peptide. We conclude that BmNPV vectors can be used successfully for expressing chromosomal genes harboring multiple introns.

Transcriptional regulation of a mouse Clara cell-specific protein (mCC10) gene by the NKx transcription factor family members thyroid transciption factor 1 and cardiac muscle-specific homeobox protein (CSX).

This report defines the elements between bp -800 and -166 that regulate the quantitative level of mouse CC10 (mCC10) transcription in the lungs. The elements in this promoter domain are the response elements for the NKx2.1 homeobox protein, thyroid transcription factor 1 (TTF1). DNase I footprint analysis identified five binding sites for TTF1 between bp -800 and - 166. These sites are located at bp -344 to -335, - 282 to -273, -268 to -263, -258 to -249, and - 199 to - 190. In addition to these enhancer elements, two TTF1 binding sites were identified in the proximal promoter region (bp - 166 to + 1), at bp -74 to -69 and -49 to -39. An identical footprint of the mCC10 promoter region was also observed with another member of the NKx family, NKx 2.5, the cardiac muscle-specific homeobox protein (CSX). Deletion and linker-scanner mutational analyses of the TTF1 binding sites in the mCC10 distal promoter region with transient cotransfection into CV1 cells with either TTF1 or CSX identified the site located between bp -282 and -273 as the major regulator of CC10 expression, with minor regulation by sites at bp -344 to -335 and -258 to -249. The importance of the NKx binding site at bp -282 to -273 was verified in vivo. Transgenic mice generated with the human growth hormone gene fused to 800 bp of the mCC10 promoter containing a mutation in the TTF1 binding site at bp -282 to -273 showed a reduction in transgene expression equal to that of the mice generated with only 166 bp of 5'-flanking DNA. This report emphasizes the importance of TTF1 or related factors as major regulators of pulmonary gene expression and demonstrates the potential of NKx proteins to bind and activate heterologous target genes.

Absence of association or genetic linkage between the angiotensin-converting-enzyme gene and left ventricular mass.

Homozygous carries of the D allele of the angiotensin-converting-enzyme (ACE) gene have been reported to be at increased risk for various cardiovascular disorders, including left ventricular hypertrophy. We investigated the potential role of the ACE gene in influencing left ventricular mass. Quantitative echocardiographic data and DNA samples were available for 2439 subjects from the Framingham Heart Study. ACE genotypes were determined by an assay based on the polymerase chain reaction. (The D allele of the ACE gene contains a deletion, whereas the I [insertion] allele does not.) Left ventricular mass and the prevalence of left ventricular hypertrophy, adjusted for clinical covariates, were analyzed according to genotype. Genetic linkage between the ACE locus and left ventricular mass was evaluated by quantitative analysis of pairs of siblings. The ACE genotype was associated neither with left ventricular mass nor with the prevalence of left ventricular hypertrophy. Mean (+/-SE) left ventricular mass (adjusted for sex) among subjects carrying the DD, DI, and II genotypes was 165+/-1.6, 165+/-1.3, and 166+/-2.0 g, respectively (P=0.90). The prevalence of left ventricular hypertrophy among the three genotype groups was 15.6 percent, 13.6 percent, and 15.6 percent, respectively (P=0.36), and the adjusted relative risk of left ventricular hypertrophy associated with the DD genotype was 1.10 (95 percent confidence interval, 0.86 to 1.19). Linkage analysis in 759 pairs of siblings using both the ACE D/I marker and a microsatellite polymorphism at the neighboring locus for the human growth hormone gene failed to support any role of ACE in influencing left ventricular mass. The ACE genotype showed no association with echocardiographically determined left ventricular mass, nor did it confer an increased risk of left ventricular hypertrophy. We found no appreciable role of the ACE gene in influencing left ventricular mass.

Nuclease sensitivity of the human growth hormone-chorionic somatomammotropin locus in pituitary and placenta suggest different mechanisms for tissue-specific regulation

The five human growth hormone (GH) and chorionic somatomammotropin (CS) genes are located at a single locus on chromosome 17. These genes share extensive nucleotide sequence similarity (∼94%) even in their flanking DNA. yet GH-N is expressed efficiently in the pituitary under the control of the pituitary-specific factor GHF-1 Pit-1 and the remaining CS-A, CS-B, CS-L and GH-V genes are transcriptionally active in the placenta. Despite this specificity in vivo, a truncated CS-A promoter can bind GHF-1 Pit-1 and allow CS-A promoter activity in pituitary cells in vitro. With a view to assessing whether the placental genes of the GH CS locus possess a different chromatin structure in the pituitary and are, thus, less transcriptionally active than the GH-N gene, we have compared the DNAase I sensitivity of GH CS in isolated pituitary and placenta cell nuclei. Our data indicate that these genes are equally sensitive in isolated human pituitary nuclei. By contrast, the CS-A, CS-B and CS-L genes were significantly ( P < 0.05) more sensitive than the GH-N gene in isolated human placenta nuclei. Although just not significant, the GH-V gene was slightly more sensitive than the GH-N gene. This pattern was also seen with nuclei from human choriocarcinoma BeWo and JEG-3 cells, which express low and extremely low levels of CS RNA, respectively, but was distinct from the pattern observed in the non placental human cervical carcinoma HeLa cell line. These data indicate that the inactivity of the CS genes in the pituitary does not correlate with a ‘closed’ chromatin structure. However, they are consistent with a role for a more ‘open’ chromatin conformation in placenta-specific expression, but not necessarily high levels of transcriptional activity.

Scanning for genes in large genomic regions: cosmid-based exon trapping of multiple exons in a single product

To facilitate the scanning of large genomic regions for the presence of exonic gene segments we have constructed a cosmid-based exon trap vector. The vector serves a dual purpose since it is also suitable for contig construction and physical mapping. The exon trap cassette of vector sCOGH1 consists of the human growth hormone gene driven by the mouse mettallothionein-1 promoter. Inserts are cloned in the multicloning site located in intron 2 of the hGH gene. The efficiency of the system is demonstrated with cosmids containing multiple exons of the Duchenne Muscular Dystrophy gene. All exons present in the inserts were successfully retrieved and no cryptic products were detected. Up to seven exons were isolated simultaneously in a single spliced product. The system has been extended by a transcription-translation-test protocol to determine the presence of large open reading frames in the trapped products, using a combination of tailed PCR primers directing protein synthesis in three different reading frames, followed by in vitro transcription-translation. Having larger stretches of coding sequence in a single exon trap product rather than small single exons greatly facilitates further analysis of potential genes and offers new possibilities for direct mutation analysis of exon trap material.

Activation of Hodgkin Cells Via the CD30 Receptor Induces Autocrine Secretion of Interleukin-6 Engaging the NF-κB Transcription Factor

The CD30 surface molecule is a recently identified member of the tumor necrosis factor/nerve growth factor receptor superfamily. Within the cytoplasmic signal transducing domain, CD30 shares no significant homology to other members of this family. Signaling events engaged via CD30 are still unknown. We here identify the NF-kappabeta transcription factor as a target of the CD30-induced signal pathway in Hodgkin's disease (HD) cells. Exposure of HD cells to CD30 ligand induces release of interleukin-6 (IL-6) that can be duplicated by cross-linking HD-cells to an agonistic anti-CD30 specific monoclonal antibody (alphaCD30), but not by cross-linking to an isotype-identical irrelevant monoclonal antibody. Cross-linking of HD cells to alphaCD30 leads to enhanced accumulation of IL-6 mRNA in a time-dependent fashion resulting from transcriptional activation of the IL-6 promoter. Transient transfection assays using a series of deleted IL-6 promoter constructs linked to the human growth hormone gene as a reporter gene furthermore indicate that transcriptional activation of the IL-6 promoter requires the presence of an intact NF-kappabeta binding site. In addition, introduction of an NF-kappabeta binding site appeared to be sufficient to confer inducibility of an heterologous promoter on activation of CD30 in HD cells. Cross-linking of CD30 promotes rapid and transient binding activity of nuclear proteins to the NF-kappabeta recognition site of the IL-6 promoter. Supershift experiments using a series of monoclonal antibodies recognizing distinct members of the NF-kappaBeta transcription factor family furthermore indicate that in CD30 cross-linked HD cells p50, p65/Rel-A, and Rel-B are present, whereas the c-rel protein is not.

Tissue-specific Expression and Dietary Regulation of Chimeric Mitochondrial 3-Hydroxy-3-methylglutaryl Coenzyme A Synthase/Human Growth Hormone Gene in Transgenic Mice

We have studied the role of the mitochondrial 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase gene in regulating ketogenesis. The gene exhibits expression in various tissues and it is regulated in a tissue-specific manner. To investigate the underlying mechanisms of this expression, we linked a 1148-base-pair portion of the mitochondrial HMG-CoA synthase promoter to the human growth hormone (hGH) gene and analyzed the expression of the hGH reporter gene in transgenic mice. mRNA levels of hGH were observed in liver, testis, ovary, stomach, colon, cecum, brown adipose tissue, spleen, adrenal glands, and mammary glands from adult mice, and also in liver and stomach, duodenum, jejunum, brown adipose tissue, and heart of suckling mice. There was no expression either in kidney or in any other nonketogenic tissue. The comparison between these data and those of the endogenous mitochondrial HMG-CoA synthase gene suggests that the 1148 base pairs of the promoter contain the elements necessary for expression in liver and testis, but an enhancer is necessary for full expression in intestine of suckling animals and that a silencer prevents expression in stomach, brown adipose tissue, spleen, adrenal glands, and mammary glands in wild type adult mice. In starvation, transgenic mice showed higher expression in liver than did wild type. Both refeeding and insulin injection reduced the expression. Fat diets, composed in each case of different fatty acids, produced similar expression levels, respectively, to those found in wild type animals, suggesting that long-, medium-, and short-chain fatty acids may exert a positive influence on the transcription rate in this 1148-base-pair portion of the promoter. The ketogenic capacity of liver and the blood ketone body levels were equal in transgenic mice and in nontransgenic mice.

Transgene expression in mammary glands of newborn rats.

Transgene expression in the mammary glands of newborn rats was studied to establish an early selection system for transgenic animals producing exogenous proteins in their milk during lactation. A fusion gene composed of the bovine alpha S1 casein gene promoter and the human growth hormone gene was microinjected into rat embryos. Transgenic lines that produced human growth hormone in their milk were established and used in this study. Immediately after birth, and without any hormone treatment, human growth hormone was found in the extracts of mammary glands from both male and female rats derived from the line secreting human growth hormone in their milk. The expression of the transgene in mammary glands of newborn rats was also detected by the presence of human growth hormone mRNA. Nontransgenic newborn rats did not express the human growth hormone gene in their mammary glands, while the mRNA for rat alpha casein, an endogenous milk protein, was found in all mammary glands from both transgenic and nontransgenic neonates. These results show that analyzing the expression of transgenes in the mammary glands of neonates is a valuable tool to select the desired transgenic animals and to shorten the selection schedules establishing the transgenic animals.

Diphtheria Toxin-mediated Ablation of Parietal Cells in the Stomach of Transgenic Mice

The self-renewing epithelial populations present in the gastric units of the mouse stomach are descended from a multipotent stem cell and undergo an orderly migration-associated differentiation followed by apoptosis. The steady state census of the three principal cell types (acid-producing parietal cells, mucus-producing pit cells, and pepsinogen and intrinsic factor-producing zymogenic cells) is accurately controlled, despite marked differences in the rates of migration of each lineage. A transgenic mouse model has been created to define functional interrelationships between the proliferation, differentiation, and death programs of these lineages. Nucleotides -1035 to +24 of the noncatalytic beta subunit gene of mouse H+/K+-ATPase were used to direct expression of an attenuated diphtheria toxin A subunit in the parietal cell lineage. These transcriptional regulatory elements are not active in members of the pit and zymogenic lineages. Stomachs, prepared from postnatal day 28-80 transgenic mice and their normal littermates, were subjected to single- and multilabel immunohistochemical studies as well as qualitative and quantitative light and electron microscopic morphologic analyses. The toxin produced complete ablation of differentiated parietal cells. Loss of parietal cells was accompanied by a 5-fold increase in the number of undifferentiated granule-free cells located in the proliferative compartment of gastric units. This amplified population of granule-free cells included the multipotent stem cell as well as committed precursors of the pit and zymogenic lineages. Loss of mature parietal cells was also associated with (i) a block in the differentiation program of the zymogenic lineage with an accumulation of pre-neck cells and a depletion of their neck and mature zymogenic cell descendants, and (ii) an approximately 2-fold amplification of pit cells. These findings are consistent with the notion that epithelial homeostasis within gastric units is maintained by instructive interactions between their different cell lineages. Unlike pit and zymogenic cells, parietal cells complete their differentiation in the gastric unit's proliferative compartment before undergoing a bipolar migration along the unit. Thus, the mature parietal cell is in a strategic position to influence decision-making among gastric epithelial cell precursors and to modulate the migration-associated terminal differentiation programs of the pit and zymogenic lineages.

Transcription of the human hepatic lipase gene is modulated by multiple negative elements in HepG2 cells

The expression of the hepatic lipase ( HL) gene is highly tissue specific. In order to identify cis-acting elements which regulate the expression of this gene in the liver, multiple deletion mutants of the 5′-flanking region of the HL gene fused to the human growth hormone gene were transfected in HepG2 cells, which normally produce HL. Transient expression assays indicated the presence of negative (at nucleotides (nt) −1576 −1342 and −623 −407 ) and positive (at nt −1862 −1576 and −50 −9 ) regulatory elements. Transfection of HeLa cells, which do not produce HL, with the same deletion constructs resulted in a similar pattern of promoter activities. However, additional negative (nt −138 −50 ) and positive (nt −407 −138 ) elements were found. DNase I footprint analysis of the proximal and distal HLpromoter sequences with HepG2 and HeLa cell nuclear extracts identified seven protected regions: A, nt −1540 −1527 ; B, −1505 −1473 ; C, −1467 −1460 ; D, −592 −577 ; E, −565 −545 ; F, −234 −220 ; and G, −70 −48 . Sites A, B, C, D and E were located within regions containing negative regulatory elements. In order to determine which nuclear factor interacts with the negative elements, sites B, D and E were mutated and the effects of mutation on competition in a gel retardation assay and on promoter activity were studied. When the binding motif for API in sites B, D and E was mutated, the specific DNA-protein complexes were not competed with the mutant oligonucleotides and promoter activity increased twofold. The magnitude of the increase is less than expected from the deletion analysis, and simultaneous mutations did not cause further increase in promoter activity, which suggests that other sites are involved in this negative modulation. These results suggest that the transcription of the HLgene in HepG2 cells is negatively modulated by multiple cis-acting negative elements and AP1-like nuclear factor may play some role in this modulation

Erythroid-specific expression of human growth hormone affects bone morphology in transgenic mice.

The regulation of bone deposition and remodeling is highly complex. To further understand the influence of growth hormone on bone deposition, several lines of transgenic mice were generated that expressed the human growth hormone gene (hGH) driven by beta-globin regulatory elements. In situ hybridization confirmed that the hGH gene in these mice was expressed in an erythroid tissue-specific manner; in the fetus hGH was expressed in the liver and in the adult mice hGH was expressed in the bone marrow. The bones of mice in two lines were visualized radiographically by mammography, and relative bone densities were measured. The transgenic mice had detectably more bone density than nontransgenic littermate controls by approximately 3 weeks of age and the relative difference in density increased with age. Histological cross-sections of the tibia showed that adult transgenic mice had increased average cortical bone thickness when compared to their controls. The hypothesis is that the local effect of hGH release from differentiating erythroid cells in the bone marrow is a major contributor to the increased bone deposition in these transgenic mice.

The Human Growth Hormone Gene Is Regulated by a Multicomponent Locus Control Region

The five-member human growth hormone (hGH)/chorionic somatomammotropin (hCS) gene cluster encodes the pituitary-specific hGH-N gene and four highly related genes (hGH-V, hCS-A, hCS-B, and hCS-L) that are expressed only in the placenta. When the hGH-N or hCS-A gene, together with all previously identified cis-acting regulatory sequences, was integrated into the mouse genome, it was expressed only sporadically and at low levels in the transgenic target organs. DNase I mapping of chromatin from expressing and nonexpressing cell types was used to identify a pituitary-specific set of DNase I-hypersensitive sites (HS) and a set of HS common to both the pituitary and placenta, centered approximately 15 and 30 kb 5' of hGH-N, respectively. When contained on a cosmid insert in their native genomic configuration, these HS consistently directed high-level, pituitary-specific expression of hGH-N in transgenic mice and appeared to define a locus control region required for hGH-N expression. Individually, each set of HS was able to mediate position-independent hGH-N expression in the pituitary but demonstrated loss of physiologic control and loss of tissue specificity. The gene-proximal set of HS contained a potent enhancer activity in the pituitary, while the more distal set appeared to function primarily to establish site-of-integration independence. These data indicate that synergistic interactions among multiple elements are required to restrict hGH-N transcription to the pituitary and generate appropriate levels of expression. In addition, these results suggest a role for both shared and unique regulatory sequences in locus control region-mediated expression of the hGH/hCS gene cluster in the pituitary and possibly the placenta.

Sequence Elements Required for Function of the MHC Class II AβGene Promoter in Murine Macrophages

We have studied the DNA sequence elements of the murine MHC class II Aβgene involved in transcriptional regulation in macrophages. For this study, the Aβpromoter was fused to the human growth hormone gene and transfected into bone marrow-derived macrophages. These macrophages were stimulated by interferon-γ (IFN-γ), after which cellular RNA was assayed for the amount of human growth hormone transcripts. A −146 to +14 fragment of the Aβpromoter was found to be sufficient to confer positive regulation by IFN-γ. Induction was suppressed by bacterial lipopolysaccharide, tumor necrosis factor (TNF-α), or maleylated bovine serum albumin. Substitution mutations were made within each of the conserved sequence elements of the promoter as well as within the spacer regions between these elements. All four conserved sequences found in class II promoters, the H, X1, X2, and Y elements, were found to be essential for promoter activation in macrophages.