Genes In Various Tissues Research Articles

Cloning multigene families with degenerate PCR primers

Multigene families often exhibit regions of highly conserved amino acid sequence that are characteristic of a given family. Degenerate PCR primers that target these motifs will selectively amplify members within that multigene family. Members of the family that are expressed in a particular tissue can be amplified from cDNA. The PCR products can be easily cloned, screened, and sequenced over a precise region to rapidly identify novel genes. PCR with degenerate primers can also be used to screen the expression of these genes in various tissues and cell types.

Modulation of Gene Expression in Autoimmune Disease and Aging by Food Restriction and Dietary Lipids

Several recent observations carried out by many investigators have offered some clues in understanding the mechanism of how food restriction (FR) acts in the prolongation of life-span, but the precise mechanisms involved in modulating the immune system have not been clearly understood. Our own ongoing studies indicate that FR may act at the molecular level and may extend the life-span by modulating functional activities of several genes in various target tissues. For instance, while cytochrome P-450 IIB1 and IIB2 expression is known to decline with age in ad libitum-fed rats, FR prevented the loss of (drug-inducible) P-450 enzymes in liver tissues. In addition, both alpha 2u-globulin and senescence marker protein 2 expressions, which are regulated by hormones, were also modulated during aging by FR in Fischer 344 male rats. In short-lived autoimmune-prone mice, both FR and omega-3 (n-3) fatty acids diet lowered the severity of autoimmune disease both in lupus-prone (NZB x NZW)F1 mice and in mice prone to develop lymphoproliferative and renal diseases, whereas saturated (n-9) and polyunsaturated (n-6) dietary lipids not only exacerbated autoimmune disease, but also significantly enhanced expression of several oncogenes in lymphoid tissues. FR and omega-3 fatty acids decreased the expression of certain oncogenes. Both FR and omega-3 fatty acids may modulate the aging and autoimmune disease processes by not only altering the fatty acid composition, membrane fluidity, and signal transduction, but also by modulating the lymphokine hormone receptors and their functions and thereby modulating expression of several genes in various tissues during the aging process.

Structural organization and differential expression of rice α-amylase genes

Rice alpha-amylases are encoded by a multigene family that has previously been classified into 5 hybridization groups. DNA sequence and Southern blot analysis identified three genes (RAmy1A, RAmy1B and RAmy1C) in Group 1 with DNA sequence identity of at least 90%. Hybridization Group 2 is represented by only one gene, RAmy3D, which is identical to a previously characterized cDNA, pOS137. RAmy3D is physically linked to the sole representative of Group 5, RAmy3E. The identity between these two genes is 81.4% in the coding region but less than 50% in the 5' and 3' flanking regions. Northern blot analysis and RNA-PCR were used to detect the expression of alpha-amylase genes in various tissues. Alpha-amylase mRNA was abundant in germinating seeds and callus. Some genes were also expressed at much lower levels in roots, young leaves and immature seeds. RAmy1A and RAmy3E were expressed in all tissues while RAmy3D was expressed in all tissues except the immature seeds. RAmy1B was weakly expressed only in callus. RAmy1A transcript was most abundant in the germinating seeds, while RAmy3D and RAmy3E transcripts were most abundant in callus and immature seeds, respectively.

Developmental regulation of the rat insulin-like growth factor I receptor gene.

We have investigated the developmental regulation of the rat insulin-like growth factor I (IGF-I) receptor gene in various tissues using a sensitive and specific solution hybridization/RNase protection assay. For this purpose we characterized rat IGF-I receptor cDNAs that were cloned from a simian virus 40-transformed rat granulosa cell cDNA library. The specific cDNA clone used in these studies encoded the putative signal peptide and the first 53 amino acids of the alpha subunit and was approximately 94% homologous to its human counterpart. IGF-I receptor gene expression was studied during the perinatal period and at various intervals until early adulthood. Overall, steady-state IGF-I receptor mRNA levels decreased dramatically during postnatal development; however, the extent of the decrease differed among the various tissues studied. In contrast to receptor mRNA levels, IGF-I mRNA levels increased in some of the same tissues. The molecular mechanisms underlying this apparent divergent transcriptional control of the IGF-I and IGF-I receptor genes warrant further study.

Regulation of vasopressin and oxytocin synthesis in anterior pituitary and peripheral tissues.

Recent studies have demonstrated the presence of immunoreactive oxytocin (OT) and vasopressin (VP), OT and VP receptors and physiological functions for these two hormones in a variety of peripheral tissues, including anterior pituitary gland. The objectives of this study were to determine if (i) OT and VP genes are expressed in rat testis and anterior pituitary gland and (ii) if osmotic stimulation known to modify the regulation of OT and VP genes in hypothalamus, would modify the expression of these genes in rat testis and anterior pituitary gland. Using oligonucleotide probes (courtesy of Drs. M. Brownstein and W. Scott Young, NIMH) corresponding to the VP gene or OT gene and specific fractions of human OT and VP genes (courtesy of Dr. J. Battey, NCI) subcloned in the pGEM-3 riboprobe system, and Northern blot and slot blot techniques, OT and VP mRNAs were found in rat testis and anterior pituitary gland. When adult male rats (SD) were either deprived of drinking water or offered 2% salt solution as a sole source of drinking fluid for 72 hrs, both OT and VP mRNA levels were increased in hypothalamus, anterior pituitary gland and testis. Our data suggest that testis and anterior pituitary gland could also be sites of synthesis of OT and VP and that the same stimulus may regulate these genes in various tissues.

Expression of growth-associated genes in various tissues of the fetal and adult rat

4F1, 2A9 and 2F1 represent three of a number of cDNA sequences which have been identified because their cognate RNAs markedly increase when quiescent cells in culture are stimulated with serum. Studies using a variety of cell culture systems have shown that the expression of these genes is modulated by various growth factors and mitogens and thus such genes are considered to be 'growth-associated.' Thus far, little information has been obtained with these in vitro systems about the function of these genes. In an attempt to begin to elucidate the role of these genes (if any) in the physiology of the normal cell, we have analyzed the levels of 4F1, 2A9 and 2F1 transcripts in a variety of differentiated organs and tissues of adult and fetal rats. Our results show that each of these growth-associated genes exhibits its own unique pattern of expression, unrelated to the proliferative activity of the tissue. These data suggest that these genes most likely do have specific functions in normal tissue in addition to their role in the induction of DNA synthesis in quiescent cells in culture.

Tissue distribution of rat S-100α and β subunit mRNAs

Using Southern blot analysis it is demonstrated that S-100α and β genes were found as single copies in the rat genome. S-100α mRNA was not found at all in rat brain during the developmental period, but it was slightly detectable in muscle and kidney. S-100β mRNA was detected in rat brain, and its mRNA level increased during the developmental period. The different localizations of the subunits may be due to the specific expression of each gene in various tissues.

Isolation of a cDNA clone encoding the leader peptide of prion protein and expression of the homologous gene in various tissues.

We have isolated a hamster cDNA clone representing the coding sequences for the entire precursor of prion protein (PrP) 27-30. This clone encodes a protein of 254 residues and contains an in-frame ATG codon 42 bases upstream from the one previously reported. Analysis of the predicted amino acid sequence suggests that the PrP precursor protein contains an amino-terminal signal sequence, and a membrane-spanning domain in the carboxyl terminus. Cleavage of the signal peptide would produce a mature protein of 232 amino acids. Sequences homologous to the hamster PrP cDNA were detected in hamster, mouse, sheep, human, and rabbit genomes. A related 2.5-kilobase transcript was present in the brain of normal and scrapie-infected rodents. Two homologous transcripts, 2.5 and 1.1 kilobases, were detected in the lung and heart of normal animals. No PrP mRNA was detected in spleen stroma, a tissue known to contain high titers of scrapie. Antisera raised to the 27- to 30-kDa polypeptide detected the PrP in both normal and infected brains but failed to detect this protein in either normal or infected spleens. Homologous mRNA species were detected in human, sheep, and rabbit brain, even though the latter is resistant to scrapie infection. Our data suggest that PrP is not a necessary component of the infectious agent.