Regulation of mouse mammary-gland gamma-glutamyltranspeptidase mRNA during pregnancy, lactation and weaning.

The level of gamma-glutamyltranspeptidase (GGT) activity and of its mRNA were determined in the mouse mammary gland during pregnancy, lactation and weaning. The GGT activity, which is very low in the virgin-mouse mammary gland (5 munits/mg of protein), increases progressively during pregnancy (3-fold), reaches its maximum at the onset of lactation (8-fold) and returns rapidly to basal level at weaning. Although no GGT-specific mRNA is detected in the virgin-mouse mammary gland, a single faint band of 2.2 kb in size is found during pregnancy. During lactation, an additional mRNA of 2.4 kb in size appears, and the level of both mRNAs is higher. This high level of mRNA persists during weaning as well. Southern-blot analysis of mouse mammary-gland DNA provides convincing evidence that there is only one gene which codes for the two mRNAs. The present study provides the first evidence for a physiological regulation of the two GGT mRNAs in the same tissue.     

Differential regulation of the accumulation of the light-harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves

The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and the light-harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de-etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40-fold, at least 200-fold, and about 25-fold, respectively, while the total RNA content per apical bud increased only 3.5-fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60-, 100-, and 200-fold, respectively. The LHCP increased steadily in concentration during de-etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24-hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far-red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far-red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady-state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.     

Induction of C-reactive protein, serum amyloid P component, and kininogens in the submandibular and lacrimal glands of rats with experimentally induced inflammation.

The mRNAs for acute-phase proteins and kininogens were found to be increased in the submandibular gland (SMG) and extraorbital and intraorbital lacrimal gland (ELG and ILG) in response to experimentally induced inflammation in rats; i.e., 24 hours after subcutaneous injection of turpentine oil, mRNAs for C-reactive protein (CRP), serum amyloid P component (SAP), and H- and T-kininogens were induced in the SMG, ELG, and ILG of rats, whereas these mRNAs were not detected in the same tissues of normal control rats. The induction of mRNAs for these inflammatory proteins by turpentine oil was preceded by a transient increase in the level of mRNA for tumor necrosis factor-alpha (TNF-alpha) at 6 hours after subcutaneous injection of the oil. This was confirmed by injection of another inflammation inducer, lipopolysaccharide (LPS), which induced the TNF-alpha mRNA in the same way at 6 hours as turpentine oil did. The up-regulation of acute-phase proteins including kininogens in the SMG, ELG, and ILG suggest the existence of a strict defense system in the exocrine glands.     

Identification and characteristics of mRNA's showing increased levels during DNA replication

A library of double-stranded cDNA was prepared using poly (A) + RNA from regenerating rat liver 20 h after partial hepatectomy. Differential screening of 350 recombinant clones with cDNA-G0 and cDNA-S identified eleven cDNA clones (pRL), the sequences of which were preferentially expressed during the DNA replication period. Levels of mRNAs complementary to these clones were 2--10-fold higher in the S-period, than in G0. Using plasmid cDNAs to different mRNA, pRL we have investigated the changes in the levels of mRNA pRL during liver regeneration. The level of mRNA mRL2 and pRL79 was increased just before DNA replication. mRNA pRL35 accumulates after partial hepatectomy with the maximum at 6 h. The augment of two other mRNA concentrations was expressed to a lesser extent. Northern-blot analysis allowed to determine the individual dual mRNAs corresponding to each of the three clones with their sizes ranging from about 1650 to 3900 bases. Three mRNAs (pRL35, 67 and 79) were shown (by hybrid-selected translation) to code for proteins of about 100, 140 and 120 kDa, respectively.     

Changes in levels of actin and tubulin mRNAs upon the lectin activation of lymphocytes.

The expression of beta-actin, gamma-actin, alpha-tubulin, and beta-tubulin mRNA during the lectin activation of human peripheral blood lymphocytes was examined with specific cDNA clones. The resting lymphocyte has a low level of both alpha- and beta-tubulin mRNAs, and these increase 10-fold after 72 h of lectin stimulation in which maximum cell transformation is achieved. Although there is a slight increase in tubulin mRNA during the first 6 h, most of the increase occurs between 6 and 24 h as the cells start to increase their RNA content and progress from G0 into G1. Both beta- and gamma-actin mRNAs are more abundant than the tubulin mRNAs in resting cells, with beta-actin mRNA being the major species. Upon activation, beta-actin mRNA increases threefold, whereas gamma-actin mRNA increases almost sixfold. Both beta- and gamma-actin mRNA are elevated 2.5-fold as early as 6 h, the gamma-actin mRNA level then increasing more than beta-actin between 6 and 24 h, resulting in the reduced beta-actin/gamma-actin mRNA ratio. The lectin-stimulated lymphocyte has a similar beta-actin/gamma-actin mRNA ratio as that of the human leukemic T-lymphoblast cell line CCRF-CEM. These increases are over and above the general increase in polyadenylated RNA content upon lectin activation. On returning to a noncycling state, the levels of these cytoskeletal mRNAs decrease. There were two beta-tubulin mRNAs present in lymphocyte cytoplasm, one of 1.8 kilobases and one of 2.8 kilobases in length. The nongrowing lymphocytes had relatively lower levels of the larger sized mRNA. Upon stimulation, the relative level of the larger mRNA was increased, and at 72 h the cells had approximately equal levels of both mRNAs as did the leukemic lymphoblasts.     

Placental, renal, and ileal sulfate transporter gene expression in mouse gestation

Sulfate is important for mammalian growth and development. During pregnancy, maternal circulating sulfate levels increase by 2-fold, enhancing sulfate availability to the fetus. We used quantitative real-time PCR to determine sulfate transporter mRNA levels during mouse gestation in three tissues: kidney and ileum, to identify transporters involved in sulfate absorption and maintaining high maternal circulating sulfate level; and placenta, to build a model of directional sulfate transport from mother to fetus. In the kidney, Slc13a1 and Slc26a1 were the most abundant sulfate transporter mRNAs, which increased by ≈2-fold at E4.5 or E6.5, whereas lower levels of Slc26a2, Slc26a6, and Slc26a7 mRNA increased by ≈3- to 6-fold from E4.5. Ileal sulfate transporter mRNA levels were not increased in gestation, but slight decreases (by ≈30-40%) were found for Slc26a3 and Slc26a6. In placentae, Slc13a4 and Slc26a2 mRNAs were most abundant, with levels increasing from E10.5 and peaking (≈8-fold) from E14.5 to E18.5, whereas Slc26a1 increased by ≈3-fold at E18.5. The spatial expression of placental mRNAs was determined by in situ hybridization showing Slc13a4 and Slc26a6 in yolk sac, Slc26a1 in spongiotrophoblasts, and Slc13a4, Slc26a2, Slc26a3, and Slc26a7 in the labyrinthine layer. Within the labyrinth, cell-specific staining revealed Slc13a4 expression in syncytiotrophoblast-II (SynT-II) and Slc26a2 in SynT-I. Together, these data show kidney Slc13a1 and Slc26a1 and placental Slc13a4 and Slc26a2 to be the most abundant sulfate transporter mRNAs in mouse gestation, which likely play important physiological roles in maintaining high maternal serum sulfate levels during pregnancy and mediating sulfate supply to the fetus.     

Organ-specific and age-dependent expression of insulin-like growth factor-I (IGF-I) mRNA variants: IGF-IA and IB mRNAs in the mouse

Insulin-like growth factor-I (IGF-I) gene generates several IGF-I mRNA variants by alternative splicing. Two promoters are present in mouse IGF-I gene. Each promoter encodes two IGF-I mRNA variants (IGF-IA and IGF-IB mRNAs). Variants differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain-coding region. Functional differences among IGF-I mRNAs, and regulatory mechanisms for alternative splicing of IGF-I mRNA are not yet known. We analyzed the expression of mouse IGF-IA and IGF-IB mRNAs using SYBR Green real-time RT-PCR. In the liver, IGF-I mRNA expression increased from 10 days of age to 45 days. In the uterus and ovary, IGF-I mRNA expression increased from 21 days of age, and then decreased at 45 days. In the kidney, IGF-I mRNA expression decreased from 10 days of age. IGF-IA mRNA levels were higher than IGF-IB mRNA levels in all organs examined. Estradiol-17beta (E2) treatment in ovariectomized mice increased uterine IGF-IA and IGF-IB mRNA levels from 3 hr after injection, and highest levels for both mRNAs were detected at 6 hr, and relative increase was greater for IGF-IB mRNA than for IGF-IA mRNA. These results suggest that expression of IGF-I mRNA variants is regulated in organ-specific and age-dependent manners, and estrogen is involved in the change of IGF-I mRNA variant expression.     

Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state

Thymidylate synthase (TS) activity is very low in resting mouse 3T6 fibroblasts but increases sharply in growth-stimulated cells at about the same time the cells enter S phase. To study the mechanism responsible for the increase in TS level, we isolated a 5-fluorodeoxyuridine (5-FdUrd)-resistant cell line (LU3-7) that overproduces TS and its mRNA about 50-100-fold. In this paper we show that the LU3-7 cells were able to rest in the G0 state of the cell cycle when maintained in medium containing 0.5% serum. When the serum concentration was increased to 10%, the resting cells reentered the cell cycle and began DNA replication about 12 hr later. TS activity remained at the resting level until DNA replication began, then increased at later times. The increase was not affected when the cells were stimulated in the presence of DNA synthesis inhibitors. The rate of synthesis of TS (as determined in a pulse-labeling experiment) remained at the resting level for the first 10 hr following stimulation, then increased 8-9-fold by 25 hr following serum stimulation. The half-life of TS in growing LU3-7 cells was measured in a pulse-chase experiment and found to be greater than 24 hr. Therefore the increase in TS activity was primarily due to an increase in the rate of synthesis of the enzyme. Since TS gene expression appears to be regulated in a similar manner in LU3-7 cells and in the parental 3T6 cells, the LU3-7 cells should be a good model system for detailed analysis of the mechanism for regulating TS gene expression in mammalian cells.     

Differential expression of the multiple forms of rat prekininogen mRNAs after acute inflammation

Responses of the rat liver prekininogen mRNAs after induction of acute inflammation were examined by blot-hybridization and S1 nuclease protection analyses with the aid of cDNA probes specific for rat kininogens. Marked changes in the relative levels of the low molecular weight (LMW) prekininogen mRNAs were observed after administration of Escherichia coli lipopolysaccharide, and the mRNA levels increased with a half-maximal dose of approximately 100 ng of lipopolysaccharide/100 g body weight. At maximum level of induction, the LMW prekininogen mRNAs comprised about 1% of total liver mRNA, thus representing a major component of the liver mRNA in the acutely inflamed rat. Differences in the inflammatory responses of various forms of the prekininogen mRNAs were then investigated by S1 nuclease protection analysis with the use of three different cDNA probes, each specific for either K-prekininogen or two types of T-prekininogens. Both of the T-prekininogen mRNAs increased progressively during the first 24 h after induction of inflammation, and at maximum level of induction, these two mRNAs increased about 10- and 13-fold over their normal level. In contrast, neither of the high molecular weight and LMW K-prekininogen mRNAs exhibited such an increase after induction of inflammation. Thus, the expressions of the rat T- and K-prekininogen mRNAs are differentially regulated in response to the induction of acute inflammation.     

Androgen-regulated ornithine decarboxylase mRNAs of mouse kidney

Ornithine decarboxylase, the first enzyme of the polyamine biosynthetic pathway, is induced by androgens in the mouse kidney. We have isolated from a kidney cDNA clone bank two plasmids, pODC1440 and pODC934 , that contain different cDNA inserts corresponding to ornithine decarboxylase mRNA. Identification was based upon the ability of plasmid-specific mRNAs to encode a 53,000-dalton polypeptide that reacts with antibody to purified mouse kidney ornithine decarboxylase. Plasmid pODC1440 hybridizes predominantly to a mRNA that is 2.1 kilobases (kb) long and is induced about 20-fold in the kidneys of female mice treated with testosterone. Plasmid pODC934 hybridizes to mRNAs of lengths 2.2 and 2.6 kb, which are induced about 8-fold by testosterone. There are probably no more than 1-2 copies of the pODC1440 -specific sequence in the mouse genome, while there may be as many as 12 copies of the pODC934 -specific sequence. That the two plasmids correspond to different ornithine decarboxylase mRNAs is suggested by two observations. First, several mouse strains express normal levels of pODC934 -specific RNA and little or no pODC1440 -specific RNA; furthermore, pODC934 -specific RNA is expressed in several tissues while pODC1440 -specific RNA is kidney-specific. Thus, androgen-mediated stimulation of kidney ornithine decarboxylase activity levels involves alterations in the concentrations of at least two distinct mRNAs.