Three novel paralogs of the rodent prolactin gene family.

The prolactin (PRL) family consists of a collection of genes expressed in the uterus, placenta and anterior pituitary. These cytokines/hormones participate in the control of maternal-fetal adaptations to pregnancy. In this report, we establish the presence of three new members of the PRL family. Novel expressed sequence tags (ESTs) with homology to PRL were isolated from embryonic and placental cDNA libraries. The cDNAs were sequenced and compared with those of other members of the PRL family. The three new cDNAs were assigned to the PRL family on the basis of sequence similarities and were referred to as PRL-like protein-J (PLP-J), PRL-like protein-K (PLP-K) and PRL-like protein-M (PLP-M). Both rat and mouse PLP-J cDNAs were identified. Rat PLP-J cDNA encodes for a predicted 211 amino acid protein containing a 29 amino acid signal peptide and two putative N-linked glycosylation sites, whereas the mouse PLP-J cDNA encodes for a 212 amino acid protein containing a 29 amino acid signal peptide with a single N-linked glycosylation site. Rat and mouse PLP-J proteins share approximately 79% and 70% nucleotide and amino acid sequence identity, respectively. A full-length rat PLP-K cDNA and a partial tentative mouse PLP-K cDNA were identified. The rat PLP-K cDNA encodes for a predicted 228 amino acid protein containing a 31 amino acid signal peptide and one putative N-linked glycosylation site; the mouse PLP-M cDNA encodes for a predicted 228 amino acid protein containing a 28 amino acid signal peptide and one putative N-linked glycosylation site. Genes for PLP-J, PLP-K and PLP-M are situated at the Prl family locus on mouse chromosome 13. PLP-J was exclusively expressed in decidual tissue from both the mouse and rat. PLP-K was expressed in trophoblast cells of the chorioallantoic placenta and showed an apparent species difference. In the mouse, virtually all trophoblast lineages expressed PLP-K, whereas in the rat, PLP-K expression was restricted to the labyrinthine trophoblast cells. Mouse PLP-M expression was restricted to the junctional zone of the chorioallantoic placenta. In summary, we have identified three new members of the rodent PRL gene family that are expressed in uterine and placental structures. Future experimentation is needed to determine the specific roles of each of these ligands in the biology of pregnancy.

A cDNA cloned from pregnant mouse uterus exhibits temporo-spatial expression and predicts a novel protein.

A cDNA was cloned from a pregnant mouse uterus cDNA library. On conceptual translation, the cDNA has one long open reading frame that predicts a novel protein of 606 amino acids. This protein is principally composed of two CUB domains and a ZP domain; motifs found in proteins implicated in egg-sperm recognition. Probes derived from the cDNA were used to conduct Northern hybridizations. The expression of this mRNA is temporal; message first appears in the uterus 6 days prior to birth, it increases each subsequent day to attain maximal levels at 3 days prior to birth and then abruptly decreases during the last 3 days of pregnancy. The expression of this mRNA is restricted; message is abundant in the uterus during late pregnancy, but it is not found in non-pregnant uterus or in a variety of adult or fetal tissues. The temporo-spatial expression of this pregnant uterus specific mRNA and the consolidation in the predicted protein of two motifs implicated in early pregnancy events suggests that the product of the gene represented by this mRNA may play an important role in events that transpire during late pregnancy.

There is an increase in expression of the cytotoxic T-lymphocyte antigen-2 alpha gene during pregnancy.

OBJECTIVE: Our purpose was to identify genes that exhibit increased expression in the uterus during pregnancy. STUDY DESIGN: A differential screen was performed against a pregnant mouse uterus complementary deoxyribonucleic acid library by use of probes derived from pregnant and nonpregnant uterus. Multiple clones related to the cytotoxic T-lymphocyte antigen-2 alpha gene were isolated. RESULTS: Northern hybridization disclosed that message is present in both the gravid and nongravid uterus, but there is a substantial increase in expression during pregnancy. Expression is not found in a variety of other fetal and adult tissues evaluated (excluding placenta). CONCLUSION: The uterus (or cells present within the uterus) constitutes a major site of in vivo expression for the cytotoxic T-lymphocyte antigen-2 alpha gene, and expression of this gene is increased during pregnancy.

An increase in expression of the lipocalin 24p3 is found in mouse uterus coincident with birth.

OBJECTIVE: To provide novel insights into the molecular events associated with parturition, a differential screen was made of a mouse uterine complementary deoxyribonucleic acid library to identify selectively expressed genes in late pregnancy. STUDY DESIGN: A differential hybridization was used to screen a mouse complementary deoxyribonucleic acid library prepared from late pregnancy uterus. A 1 kb clone was isolated that was subsequently identified as 24p3, a member of the lipocalin family. By use of radiolabeled complementary deoxyribonucleic acid probes prepared from this clone Northern hybridizations were conducted against total ribonucleic acid purified from mouse uterus collected during late pregnancy and the first week after birth and a variety of other mouse tissues to determine whether this gene is selectively expressed in the uterus coincident with parturition. RESULTS: Low levels of message for the 24p3 gene could be detected in uterine ribonucleic acid, but there was a massive increase in the level of message for this gene on the days surrounding birth. Northern hybridizations conducted against additional tissues collected from both pregnant and nonpregnant mice did not detect message to a similar degree as found in the uterus. CONCLUSIONS: The uterus constitutes a major site of expression of this gene, particularly near birth. The expression of this gene coincident with birth suggests a potential physiologic role of the neutrophil with the induction of labor.

Selenoprotein P expression in liver, uterus and placenta during late pregnancy.

To identify genes that exhibit increased expression in the placenta during late pregnancy, the technique of differential cDNA library screening was used to isolate a clone subsequently identified as the 3' untranslated region of the mouse selenoprotein p gene. Random primed radiolabelled cDNA probes were constructed from this clone and these probes were used to conduct Northern hybridizations against total RNA purified from mouse placenta, liver (maternal and fetal) and uterus collected sequentially during the latter third of pregnancy. Signal is present in the placenta and beginning 4 days before birth, the level of message increases, reaching maximal levels at term. The level of expression in the placenta at maximum is approximately 25 per cent of that observed in adult liver. In liver obtained from pregnant females, the level of message is increased compared to nonpregnant adults, but returns to normal shortly after birth. Message is also found in the fetal liver beginning at 4 days before birth and exhibits a pattern of expression similar to the placenta. The similarity of expression observed in fetal liver and placenta suggests a coordinated regulation of expression of this gene in these tissues. There is a minimal amount of signal present in the uterus and the expression does not appear to vary. We speculate that selenoprotein p may play a role in the transplacental transport of selenium to the fetus during late pregnancy.

Transferrin gene expression in maternal liver, fetal liver and placenta during pregnancy in the mouse.

Two clones that are homologous to the mouse liver transferrin gene were isolated from a differential screen performed on a mouse cDNA library constructed from placenta. Using an insert derived from the larger of these clones as a template for the generation of random primed cDNA probes, northern blots were conducted against total RNA collected sequentially from placenta (7 days before birth to birth), maternal liver (7 days before birth to birth) and fetal liver (5 days before birth to birth). An approximately 2.3 kb message was detected in all three tissues which was upregulated in late gestation. Message was very abundant in both maternal and fetal liver, and present, but weak, in placenta. The clones were partially sequenced and both clones contain sequence that is identical to mouse liver transferrin. The data presented demonstrate an increase in mRNA transferrin in late gestation in maternal and fetal liver. Additionally, the placenta expresses a gene homologous to liver transferrin and it also is upregulated in late gestation.

Kidney androgen-regulated protein gene is expressed in the uterus during late pregnancy.

Kidney androgen-regulated protein (KAP) is a unique protein of unknown function that is transcriptionally induced by sex steroids. KAP is thought to be predominantly a kidney-specific gene. After conducting a differential screen of a mouse uterus cDNA library, a clone was identified that is identical to KAP. Using this cDNA to generate radiolabeled cRNA probes, Northern blots were conducted against the following tissues collected sequentially during the latter third of pregnancy: kidney, uterus and placenta. Abundant message was present in all samples of the kidney tested and there was a slight, but apparent, increase (1.5-fold) in expression during the period surrounding birth. Message is also present in the uterus, at levels comparable to the kidney, but expression occurs only during the period surrounding birth. Message is not present in the uterus at any other time. Message is also not detected in the placenta or in several other tissues tested. In addition to the kidney, KAP gene is also transcribed at equivalent levels in the uterus. Unlike the kidney, expression in the uterus is limited to the perinatal period.

Developmental and tissue-specific expression of nuclear proteins that bind the regulatory element of the major histocompatibility complex class I gene.

Expression of MHC class I genes varies according to developmental stage and type of tissues. To study the basis of class I gene regulation in tissues in vivo, we examined binding of nuclear proteins to the conserved cis sequence of the murine H-2 gene, class I regulatory element (CRE), which contains two independent factor-binding sites, region I and region II. In gel mobility shift analyses we found that extracts from adult tissues that express class I genes, such as spleen and liver, had binding activity to region I. In contrast, extracts from brain, which does not express class I genes, did not show region I binding activity. In addition, fetal tissues that express class I gene at very low levels, also did not reveal region I binding activity. Binding activity to region I became detectable during the neonatal period when class I gene expression sharply increases. Most of these tissues showed binding activity to region II, irrespective of class I gene expression. Although region II contained a sequence similar to the AP-1 recognition site, AP-1 was not responsible for the region II binding activity detected in this work. These results illustrate a correlation between region I binding activity and developmental and tissue-specific expression of MHC class I genes. The CRE exerts an enhancer-like activity in cultured fibroblasts. We evaluated the significance of each factor binding to CRE. Single 2-bp mutations were introduced into the CRE by site-directed mutagenesis and the ability of each mutant to elicit the enhancer activity was tested in transient CAT assays. A mutation that eliminated region I protein binding greatly impaired enhancer activity. A mutation that eliminated region II binding also caused a lesser but measurable effect. We conclude that region I and region II are both capable of enhancing transcription of the class I gene. These results indicate that in vivo regulation of MHC class I gene expression is mediated by binding of trans-acting factors to the CRE.

Neonatal induction of a nuclear protein that binds to the c-fos enhancer.

The expression of the c-fos gene is transiently induced at birth in most organs in the mouse. To study the basis of this induction we searched for a nuclear factor that binds to the 5' regulatory region of the c-fos gene. Gel mobility shift assays with tissue extracts revealed fast (band I) and slow (band III) migrating bands, which represent factor binding to the c-fos enhancer, termed the serum response element (SRE). Neonatal extracts preferentially elicited band I, with low or undetectable levels of band III, whereas fetal and adult extracts generated predominantly band III, with reduced levels of band I. These results indicate that the SRE-binding activity changes during perinatal development and that the appearance of band I, which coincides with diminution of band III, correlates with neonatal c-fos induction. Methylation interference and competition analyses showed that the neonatal factor (band I) binds to the SRE at a site different from the adult factor (band III). DNA-binding activity of the adult factor, but not the neonatal factor, was sensitive to phosphatase treatment. Furthermore, the adult factor, but not the neonatal factor, shared antigenic specificity with the human serum response factor (SRF) that is expressed in cultured cells irrespective of c-fos gene induction. We conclude that band I in neonates represents a SRE-binding factor that is distinct from the SRF, which may be responsible for the neonatal induction of the c-fos gene. The band III factor was indistinguishable from the SRF in all criteria tested.

Expression of ets genes in mouse thymocyte subsets and T cells.

The cellular ets genes (ets-1, ets-2, and erg) have been identified by their sequence similarity with the v-ets oncogene of the avian erythroblastosis virus, E26. Products of the ets-2 gene have been detected in a wide range of normal mouse tissues and their expression appears to be associated with cell proliferation in regenerating liver. In contrast, the ets-1 gene was previously shown to be more highly expressed in the mouse thymus than in other tissues. Because the thymic tissue contains various subsets of cells in different stages of proliferation and maturation, we have examined ets gene expression in fetal thymocytes from different stages of development, in isolated subsets of adult thymocytes, and in peripheral T lymphocytes. Expression of the ets-1 gene was first detected at day 18 in fetal thymocytes, corresponding to the first appearance of CD4+ (CD4+, CD8-) thymocytes, and reaches maximal/plateau levels of expression in the thymus at 1 to 2 days after birth. The ets-2 gene expression is detected at least 1 day earlier, coinciding with the presence of both double-positive (CD4+, CD8+) and double-negative (CD4-, CD8-) blast thymocytes and reaches maximal/plateau levels 1 day before birth. In the adult thymus, ets-1 and ets-2 mRNA expression is 10- to 8-fold higher respectively in the CD4+ subset than in the other subsets examined. Higher levels of p55 ets-1 protein were also shown to exist in the CD4+ subset. Because the CD4+ thymic subset is the pool from which the CD4+ peripheral, helper/inducer T cells are derived, the ets gene expression was examined in lymph node T cells. Both the CD4+ and the CD8+ T cells subsets had lower ets RNA levels than the CD4+ thymocytes. These results suggest that ets-2 and more particularly ets-1 gene products play an important role in T cell development and differentiation and are not simply associated with proliferating cells, which are observed at a higher frequency in fetal thymocytes, or dull Ly-1 (low CD5+), and double-negative (CD4-, CD8-) adult thymocytes. Selectively enhanced expression of ets-1 gene may be observed in thymic CD4+ thymocytes because these cells have uniquely encountered MHC class II or other Ag in the thymic environment. These cells may have been subsequently stimulated to activate the ets genes in conjunction with their differentiation of helper/inducer function(s) and expression of mature TCR.(ABSTRACT TRUNCATED AT 400 WORDS)

c-fos antisense RNA blocks expression of c-fos gene in F9 embryonal carcinoma cells.

To study the function of proto-oncogene c-fos, we prepared an antisense plasmid that expresses in mammalian cells c-fos antisense RNA which is complementary to the endogenous c-fos mRNA. Upon transfection into undifferentiated F9 EC cells, the antisense plasmid directed constitutive expression of a large amount of c-fos antisense RNA. These cells were very low in the basal level of c-fos message and were unable to induce c-fos message when stimulated with interferon or phorbol ester. The failure to induce c-fos message led to the blockade of c-fos protein expression in these cells. Thus, these cells represented a c-fos defective phenotype. The blockade of c-fos gene expression seen in antisense-cells could be caused by rapid degradation of the c-fos message, since c-fos mRNA expression was rescued in these cells when treated with protein synthesis inhibitor, cycloheximide. We found that expression of c-myc gene was down-regulated in c-fos antisense-cells: Although control undifferentiated F9 cells constitutively expressed a high level of c-myc message, the antisense cells had a much lower amount of c-myc mRNA. Since p53 and heat shock gene 70 were expressed at comparable levels in control and antisense cells, c-myc gene expression appears to be regulated by c-fos gene in F9 EC cells. Lastly, these antisense cells grew as rapidly as control F9 cells and underwent differentiation after retinoic acid treatment, indicating that c-fos expression is not a prerequisite for differentiation of F9 cells.

A burst of c-fos gene expression in the mouse occurs at birth.

Expression of the c-fos gene during murine perinatal development was studied. Before birth, all eight of the prenatal organs tested expressed undetectable or low levels of c-fos mRNA. On the day of birth, there occurred a 10- to 100-fold increase in the level of c-fos message in all of these organs. The expression was transient, in that 1 day after birth, the level of c-fos mRNA precipitously dropped. The c-fos gene expression at birth is unrelated to the expression of the c-myc gene and major histocompatibility complex class I genes, which display distinct kinetics during the perinatal development. The c-fos gene was also expressed locally and transiently in the gravid uterus 1 to 2 days prior to delivery. These results indicate that an event associated with birth induced c-fos gene expression in the mother and newborn.

Antenatal prediction of sex.

Current recommendations suggest that prospective parents be informed of the fetal sex following an amniocentesis--if the parents express a desire for that information. Genotype does not in all cases accurately predict the appropriate sex of rearing. A case is discussed involving an infant with ambiguous genitalia who prior to birth had been designated a male, based on genotype obtained following amniocentesis. Caution is urged in predicting sex antenatally.

Rhabdomyolysis and myoglobinemia in neonates.

Serial myoglobin determinations were made in 20 neonates during the first week of life to determine whether birth asphyxia results in ischemic damage to muscle with the subsequent pathologic release of myoglobin. Serum myoglobin values were significantly elevated in asphyxiated infants compared with control infants. High myoglobin values correlated with a longer duration of oliguria in the neonatal intensive care unit population. The value of urine dipstick testing for myoglobinuria screening was also evaluated. Infants with elevated myoglobin values were more likely to have a strongly positive urine dipstick for occult blood in the first 48 hours of life. These data suggest that ischemic damage to muscle with pathologic release of myoglobin occurs in the neonatal period and that urine dipstick testing provides a reasonable screening examination for myoglobinuria.

Postconceptional age and gentamicin elimination half-life.

We examined the correlation between the elimination half-life of gentamicin and postnatal or postconceptional age in 104 infants greater than 24 weeks to less than or equal to 48 weeks postconceptional age. A weak correlation was observed between half-life and postnatal age (R = -0.353). A better correlation existed between postconceptional age and half-life (R = -0.572). A plot of the log of the mean half-life against postconceptional age was virtually linear. We conclude that postconceptional age is a major determinant of an infant's ability to excrete gentamicin, and speculate that the relationship between half-life and decreasing postconceptional age is exponential.