Cloning and mapping of 5' exons from the gene encoding chicken beta nerve growth factor.

An NGF cDNA containing the 5' exons of the nerve growth factor (NGF) messenger was obtained from chicken heart mRNA using the anchored polymerase chain reaction technique. Alignment of the chicken with the corresponding murine and human sequences reveals interspecies similarities. A sequence corresponding to an exon found only in the NGF messenger, which is abundant in the submaxillary gland of the male mouse, is present in the chicken NGF cDNA. The first non-coding exons of the NGF gene are much less conserved between chicken and mouse or human than the region of the last exon encoding the mature protein. After the cloning of the chicken NGF gene from a cosmid library, the chicken NGF exons have been located within 20 kb of DNA. The chicken NGF gene is therefore shorter than its murine counterpart which spans more than 43 kb. Furthermore, the organization of the chicken and murine NGF genes markedly differs in their 5' portion.     

Human growth hormone DNA sequence and mRNA structure: possible alternative splicing.

We have determined the complete sequence of the human growth hormone (hGH) gene and the position of the mature 5' end of the hGH mRNA within the sequence. Comparison of this sequence with that of a cloned hGH cDNA shows that the gene is interrupted by four intervening sequences. S1 mapping shows that one of these intervening sequences has two different 3' splice sites. These alternate splicing pathways generate hGH peptides of different sizes which are found in normal pituitaries. Comparison of sequences near the 5' end of the hGH mRNA with a similar region of the alpha subunit of the human glycoprotein hormones reveals an unexpected region of homology between these otherwise unrelated peptide hormones.     

Accurate spatial and temporal transgene expression driven by a 3.8-kilobase promoter of the bovine β-casein gene in the lactating mouse mammary gland

The spatial, temporal, and hormonal pattern of expression of the β-casein gene is highly regulated and confined to the epithelial cells of the lactating mammary gland. Previous studies have shown that 1.7 kb of the bovine β-casein promoter were able to drive cell-specific and hormone-dependent expression to a mouse mammary cell line but failed to induce accurate expression to the mammary gland of transgenic mice. We investigated here the ability of 3.8 kb of the bovine β-casein gene promoter to drive the expression of the human growth hormone (hGH) gene in transgenic mice. A Northern blot analysis using total RNA obtained from different tissues of lactating and nonlactating females revealed the presence of hGH mRNA only in the mammary gland of lactating females. hGH mRNA was not detectable in the mammary gland of virgin females or males. A developmental analysis showed that hGH mRNA only peaked on parturition, resembling more closely the bovine β-casein temporal expression pattern rather than the murine. In situ hibridization studies performed on mammary gland sections showed that the cellular pattern of hGH expression was homogeneous in all lobules from heterozygous and homozygous transgenic mice. Silver grain counts on the tissue sections highly correlated with the hGH contents in the milk determined by radioimmunoassay (r = 0.996). Thus 3.8 kb of the bovine β-casein promoter direct a high-level expression of a reporter gene to the lactating mammary gland of transgenic mice in a tissue-specific and developmentally regulated manner. Mol. Reprod. Dev. 49:236–245, 1998. © 1998 Wiley-Liss, Inc.     

Normalization of growth in hypophysectomized mice using hydrodynamic transfer of the human growth hormone gene

Nonviral gene transfer was investigated as a potential treatment of growth hormone deficiency (GHD) using hypophysectomized mice as a model. After a single hydrodynamic administration of naked plasmid DNA containing the human growth hormone (hGH) gene controlled by an ubiquitin promoter, sustained elevation of circulating hGH was observed the entire observation period (68 days), with a concomitant normalization of circulating insulin-like growth factor I (IGF-I) and IGF-binding protein-3. Furthermore, longitudinal growth was corrected in terms of normalization of tibia length, tail length, and body weight gain. Liver, spleen, and lung weights were normalized, whereas heart weight was normalized partly. hGH mRNA was expressed exclusively in liver tissue. In conclusion, we showed that nonviral hGH gene transfer normalizes longitudinal growth in hypophysectomized mice, indicating that this method potentially could be relevant as a new therapeutic tool in the clinical handling of GHD.     

Tissue-specific, developmental, hormonal, and dietary regulation of rat phosphoenolpyruvate carboxykinase-human growth hormone fusion genes in transgenic mice.

The cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene is expressed in multiple tissues and is regulated in a complex tissue-specific manner. To map the cis-acting DNA elements that direct this tissue-specific expression, we made transgenic mice containing truncated PEPCK-human growth hormone (hGH) fusion genes. The transgenes contained PEPCK promoter fragments with 5' endpoints at -2088, -888, -600, -402, and -207 bp, while the 3' endpoint was at +69 bp. Immunohistochemical analysis showed that the -2088 transgene was expressed in the correct cell types (hepatocytes, proximal tubular epithelium of the kidney, villar epithelium of the small intestine, epithelium of the colon, smooth muscle of the vagina and lungs, ductal epithelium of the sublingual gland, and white and brown adipocytes). Solution hybridization of hGH mRNA expressed from the transgenes indicated that white and brown fat-specific elements are located distally (-2088 to -888 bp) and that liver-, gut-, and kidney-specific elements are located proximally (-600 to +69 bp). However, elements outside of the region tested are necessary for the correct developmental pattern and level of PEPCK expression in kidney. Both the -2088 and -402 transgenes responded in a tissue-specific manner to dietary stimuli, and the -2088 transgene responded to glucocorticoid stimuli. Thus, different tissues utilize distinct cell-specific cis-acting elements to direct and regulate the PEPCK gene.     

在给利血平后的恢复中小鼠颌下腺的去甲肾上腺素含量与神经生长因子水平的关系

给ICR/JCL品系的成年小鼠1次注射利血平(2mg/kg体重)后24小时,其颌下腺(SMG)的去甲肾上腺素(NA)贮存能够被耗竭。在注射后第12天,NA 含量恢复到利血平处理以前的水平。在恢复过程中,雄性腺体的 NA 含量均为雌性的2倍左右。切断颈上神经节(SCG)的节前神经,雌雄腺体的 NA 含量都下降至对照侧的80%左右。消除节前神经的影响以后,在 NA 的恢复过程中,雄性腺体的 NA 含量仍然是雌性的2倍左右。利用神经生长因子(NGF)生物测定法,我们测得本品系成年雄性小鼠 SMG 的 NGF 含量为雌性小鼠的94倍左右。正如外源性 NGF 能选择性地提高交感神经元内的酪氨酸羟化酶活力与 SMG 的 NA 含量一样,在利血平处理后的恢复过程中,雄性腺体比雌性腺体具有较高的 NA 含量可能是由于内源性 NGF 对交感神经元内酪氨酸羟化酶产生效应的结果。     

Androgen dependence of specific kallikrein gene family members expressed in rat prostate

We have used oligonucleotide probes specific for members of the rat kallikrein/tonin gene family (PS, S1, S2, S3, K1, and P1) to establish which arginyl esteropeptidase (kallikrein-like) genes are expressed in the prostate. We have also compared the expression and androgen dependence of these genes in prostate, submaxillary gland (SMG) and kidney. Only S3 (tonin-like) and P1 (kallikrein-like) are expressed in the prostate, with S3 very much more abundant. Prostatic S3 mRNA disappears after 8 days castration and is restored to intact levels by dihydrotestosterone (DHT) but not estradiol benzoate (EB) for 8 days. Prostate P1 mRNA levels were similarly but not identically affected. All six genes are expressed in the SMG, with PS (true kallikrein) the most abundant. Levels of PS mRNA in SMG are unaffected by castration, DHT, or EB treatment, although mRNA levels of other kallikrein-like (S1, K1, and P1), tonin (S2), and tonin-like (S3) genes fall 40-60% after castration, and are unaffected or partially restored by DHT and/or EB administration. Only PS and K1 are expressed in the kidney, at much lower levels than in the SMG and unaffected by castration or steroids. These studies thus confirm and extend the concept of tissue specificity of arginyl esteropeptidase gene expression, and further demonstrate that the same gene(s) is differentially regulated by androgens in the rat prostate, SMG, and kidney.     

Kidney and submaxillary gland renins are encoded by two non-allelic genes in Swiss mice.

Two distinct phenotypic groups of inbred strains of mice, with different amounts of submaxillary gland (SMG) renin have been described. We have previously shown that strains with high levels of SMG renin, such as Swiss or AKR mice, have two renin genes, Rn1 and Rn2, per haploid genome, while strains with low levels of SMG, such as BALB/c or C57Bl/6, have only one renin gene. We now report the molecular cloning of cDNA copies of Swiss mouse kidney renin mRNA and present nucleotide sequence data of the recombinant clones. Comparison of these sequences with the sequence of Swiss mouse SMG renin mRNA we have previously reported, demonstrates that Swiss mice express the two non-allelic genes, Rn1 and Rn2.     

Nerve growth factor receptors in the peripubertal rat ovary.

We previously demonstrated that the immature rat ovary synthesizes nerve growth factor (NGF), and that interference of NGF actions by immunoneutralization during neonatal life prevents development of the ovarian sympathetic innervation and delays follicular maturation. Since the actions of NGF are exerted via binding to specific cell surface receptors, the present study was undertaken to define and characterize the presence of NGF receptors (NGFrec) in the developing rat ovary. NGF interacts with two classes of NGFrec. The most abundant is a low affinity form expressed in the central nervous system and peripheral tissues. This receptor is encoded by a single 3.8-kilobase mRNA species. Cross-linking of [125I]NGF to ovarian membranes followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography showed the presence of a approximately 90-kilodalton molecular species which corresponds in size to the predominant NGF receptor species cross-linked to its ligand. While ovarian NGFrec may be of neuronal origin and reach the gland exclusively by anterograde axonal transport, RNA blot hybridization demonstrated that the ovary expresses the NGFrec mRNA species that encodes the low affinity NGF receptor and, thus, implicated the ovary itself as a site of NGFrec synthesis. NGFrec mRNA levels decreased abruptly after the first ovulation, suggesting that NGFrec may be synthesized in growing follicles and that this capacity is lost after follicular rupture and luteinization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and expression of the chicken beta nerve growth factor gene.

The 3' exon of the chicken beta nerve growth factor (NGF) gene was isolated by the use of a murine cDNA probe. DNA sequence analysis of the clone suggests a mature chicken NGF protein of 118 amino acids, showing approximately 85% homology to mouse and human NGF. In addition to this conservation of the mature NGF, parts of the propeptide and the untranslated 3' end of the NGF gene are also highly homologous in chicken, human and mouse. Therefore, these sequences probably subserve important functions. Expression of NGF mRNA in various chicken tissues was examined by RNA blot analysis with a chicken NGF probe. A single mRNA of 1.3 kb was detected at high levels in heart and brain of 10-week-old roosters, and, at lower levels in spleen, liver and skeletal muscle. These data suggest a correlation between NGF expression and the density of sympathetic innervation in peripheral organs, in analogy with findings for mammalian tissues. In the adult avian brain, NGF mRNA is found at higher concentration in the optic tectum and cerebellum than in the cortex and hippocampus. This pattern of NGF expression differs from that previously described for the rat brain. During late stages of development (day 18), NGF mRNA was expressed both in heart and brain of embryos but at lower levels than in the adult.