Withdrawn: Discovering Genes Essential to the Hypothalamic Regulation of Human Reproduction Using a Human Disease Model: Adjusting to Life in the "-Omics" Era.

The neuroendocrine regulation of reproduction is an intricate process requiring the exquisite coordination of an assortment of cellular networks, all converging on the GnRH neurons. These neurons have a complex life history, migrating mainly from the olfactory placode into the hypothalamus, where GnRH is secreted and acts as the master regulator of the hypothalamic-pituitary-gonadal axis. Much of what we know about the biology of the GnRH neurons has been aided by discoveries made using the human disease model of isolated GnRH deficiency (IGD), a family of rare Mendelian disorders that share a common failure of secretion and/or action of GnRH causing hypogonadotropic hypogonadism. Over the last 30 years, research groups around the world have been investigating the genetic basis of IGD using different strategies based on complex cases that harbor structural abnormalities or single pleiotropic genes, endogamous pedigrees, candidate gene approaches as well as pathway gene analyses. Although such traditional approaches, based on well-validated tools, have been critical to establish the field, new strategies, such as next-generation sequencing, are now providing speed and robustness, but also revealing a surprising number of variants in known IGD genes in both patients and healthy controls. Thus, before the field moves forward with new genetic tools and continues discovery efforts, we must reassess what we know about IGD genetics and prepare to hold our work to a different standard. The purpose of this review is to: 1) look back at the strategies used to discover the "known" genes implicated in the rare forms of IGD; 2) examine the strengths and weaknesses of the methodologies used to validate genetic variation; 3)substantiate the role of known genes in the pathophysiology of the disease; and 4) project forward as we embark upon a widening use of these new and powerful technologies for gene discovery. (Endocrine Reviews 36: 603-621, 2015).

Discovering Genes Essential to the Hypothalamic Regulation of Human Reproduction Using a Human Disease Model: Adjusting to Life in the "-Omics" Era.

The neuroendocrine regulation of reproduction is an intricate process requiring the exquisite coordination of an assortment of cellular networks, all converging on the GnRH neurons. These neurons have a complex life history, migrating mainly from the olfactory placode into the hypothalamus, where GnRH is secreted and acts as the master regulator of the hypothalamic-pituitary-gonadal axis. Much of what we know about the biology of the GnRH neurons has been aided by discoveries made using the human disease model of isolated GnRH deficiency (IGD), a family of rare Mendelian disorders that share a common failure of secretion and/or action of GnRH causing hypogonadotropic hypogonadism. Over the last 30 years, research groups around the world have been investigating the genetic basis of IGD using different strategies based on complex cases that harbor structural abnormalities or single pleiotropic genes, endogamous pedigrees, candidate gene approaches as well as pathway gene analyses. Although such traditional approaches, based on well-validated tools, have been critical to establish the field, new strategies, such as next-generation sequencing, are now providing speed and robustness, but also revealing a surprising number of variants in known IGD genes in both patients and healthy controls. Thus, before the field moves forward with new genetic tools and continues discovery efforts, we must reassess what we know about IGD genetics and prepare to hold our work to a different standard. The purpose of this review is to: 1) look back at the strategies used to discover the "known" genes implicated in the rare forms of IGD; 2) examine the strengths and weaknesses of the methodologies used to validate genetic variation; 3) substantiate the role of known genes in the pathophysiology of the disease; and 4) project forward as we embark upon a widening use of these new and powerful technologies for gene discovery.

Compensatory Increase in Ovarian Aromatase in Older Regularly Cycling Women.

CONTEXT: Serum estradiol (E2) levels are preserved in older reproductive-aged women with regular menstrual cycles despite declining ovarian function. OBJECTIVE: The objective of the study was to determine whether increased granulosa cell aromatase expression and activity account for preservation of E2 levels in older, regularly cycling women. DESIGN: The protocol included daily blood sampling and dominant follicle aspirations at an academic medical center during a natural menstrual cycle. SUBJECTS: Healthy, regularly cycling older (36-45 y; n = 13) and younger (22-34 y; n = 14) women participated in the study. MAIN OUTCOME MEASURES: Hormone levels were measured in peripheral blood and follicular fluid aspirates and granulosa cell CYP19A1 (aromatase) and FSH-R mRNA expression were determined. RESULTS: Older women had higher FSH levels than younger women during the early follicular phase with similar E2 but lower inhibin B and antimullerian hormone levels. Late follicular phase serum E2 did not differ between the two groups. Follicular fluid E2 [older (O) = 960.0 [interquartile range (IQR) 765.0-1419.0]; younger (Y) = 994.5 [647.3-1426.5] ng/mL, P = 1.0], estrone (O = 39.6 [29.5-54.1]; Y = 28.8 [22.5-42.1] ng/mL, P = 0.3), and the E2 to testosterone (T) ratio (O = 109.0 ± 41.9; Y = 83.0 ± 18.6, P = .50) were preserved in older women. Granulosa cell CYP19A1 expression was increased 3-fold in older compared with younger women (P < .001), with no difference in FSH-R expression. CONCLUSIONS: Ovarian aromatase expression increases with age in regularly cycling women. Thus, up-regulation of aromatase activity appears to compensate for the known age-related decrease in granulosa cell number in the dominant follicle to maintain ovarian estrogen production in older premenopausal women.

Acute mood but not cognitive improvements following administration of a single multivitamin and mineral supplement in healthy women aged 50 and above: a randomised controlled trial.

A number of randomised controlled trials have indicated that multivitamin/mineral supplementation for a period of 4 weeks or greater can enhance mood and cognition. To date, no studies have investigated whether a single multivitamin dose can benefit mental function in older adults. This study investigated the acute effects of a single multivitamin and mineral and herbal (MVMH) supplement versus placebo on self ratings of mood and the performance of an effortful computerised cognitive battery in a sample of 76 healthy women aged 50-75 years. Mood was assessed using the depression anxiety stress scale (DASS), state trait anxiety inventory-state anxiety scale and visual analogue scales (VAS). Mood was rated at 1 h post supplementation and again after the competition of the cognitive assessments at 2 h post supplementation. It was demonstrated that the MVMH supplement improved overall DASS mood ratings; however, the most prominent effects appeared to be a reduction in ratings of perceived mental stress. These findings were confirmed using visual analogue scales, with these measures also demonstrating MVMH-related increased ratings of calmness. There were no benefits of the MVMH to mood ratings of depression and performance was not enhanced on the cognitive battery. Supplementation with a single multivitamin, mineral and herbal supplement reduces stress several hours after intake in healthy older people.

Evidence that increased ovarian aromatase activity and expression account for higher estradiol levels in African American compared with Caucasian women.

CONTEXT: Serum estradiol levels are significantly higher across the menstrual cycle in African American (AAW) compared with Caucasian women (CW) in the presence of similar FSH levels, yet the mechanism underlying this disparity is unknown. OBJECTIVE: The objective of the study was to determine whether higher estradiol levels in AAW are due to increased granulosa cell aromatase mRNA expression and activity. DESIGN: The design of the study included daily blood sampling and dominant follicle aspirations at an academic medical center during a natural menstrual cycle. SUBJECTS: Healthy, normal cycling AAW (n = 15) and CW (n = 14) aged 19-34 years participated in the study. MAIN OUTCOME MEASURES: Hormone levels in peripheral blood and follicular fluid (FF) aspirates and aromatase and FSH receptor mRNA expression in granulosa cells were measured. RESULTS: AAW had higher FF estradiol [1713.0 (1144.5-2032.5) vs 994.5 (647.3-1426.5) ng/mL; median (interquartile range); P < .001] and estrone [76.9 (36.6-173.4) vs 28.8 (22.5-42.1) ng/mL; P < .001] levels than CW, independent of follicle size. AAW also had lower FF androstenedione to estrone (7 ± 1.8 vs 15.8 ± 4.1; mean ± SE; P = .04) and T to estradiol (0.01 ± 0.002 vs 0.02 ± 0.005; P = .03) ratios, indicating enhanced ovarian aromatase activity. There was a 5-fold increase in granulosa cell aromatase mRNA expression in AAW compared with CW (P < .001) with no difference in expression of FSH receptor. FSH, inhibin A, inhibin B, and AMH levels were not different in AAW and CW. CONCLUSIONS: Increased ovarian aromatase mRNA expression, higher FF estradiol levels, and decreased FF androgen to estrogen ratios in AAW compared with CW provide compelling evidence that racial differences in ovarian aromatase activity contribute to higher levels of estradiol in AAW across the menstrual cycle. The absence of differences in FSH, FSH receptor expression, and AMH suggest that population-specific genetic variation in CYP19, the gene encoding aromatase, or in factors affecting its expression should be sought.

The effects of multivitamin supplementation on mood and general well-being in healthy young adults. A laboratory and at-home mobile phone assessment.

Previous research has suggested that multivitamin (MV) supplementation may be associated with beneficial effects for mood and general well-being, although treatment durations have typically been less than 90 days, samples have often been restricted to males only and acute effects have not been adequately differentiated from chronic effects. In the current study a MV supplement containing high levels of B-vitamins was administered daily to 138 healthy young adult participants between the ages of 20 and 50 years over a 16-week period. Chronic mood measures (GHQ-28, POMS, Chalder fatigue, PILL, Bond-Lader and custom visual analogue scales) were administered pre-dose at baseline, 8- and 16-weeks. Changes in Bond-Lader and VAS in response to a multi-tasking framework (MTF) were also assessed at 8- and 16-weeks. For a subset of participants, at-home mobile-phone assessments of mood were assessed on a weekly basis using Bond-Lader and VAS. No significant treatment effects were found for any chronic laboratory mood measures. In response to the MTF, a significant treatment x time interaction was found for STAI-S, with a trend towards a greater increase in stress ratings for male participants in the MV group at 16 weeks. However, this finding may have been attributable to a larger proportion of students in the male MV group. In contrast, at-home mobile-phone assessments, where assessments were conducted post-dose, revealed significantly reduced stress, physical fatigue and anxiety in the MV group in comparison to placebo across a number of time points. Further research using both acute and chronic dosing regimens are required in order to properly differentiate these effects.

Sex differences in juvenile mouse social behavior are influenced by sex chromosomes and social context.

Play behavior in juvenile primates, rats and other species is sexually dimorphic, with males showing more play than females. In mice, sex differences in juvenile play have only been examined in out-bred CD-1 mice. In this strain, contrary to other animals, male mice display less play soliciting than females. Using an established same-sex dyadic interaction test, we examined play in in-bred C57BL/6J (B6) 21-day-old mice. When paired with non-siblings, males tended to be more social than females, spending more time exploring the test cage. Females displayed significantly more anogenital sniffing and solicited play more frequently than did males. To determine if the origin of the sex difference was sex chromosome genes or gonadal sex, next we used the four core genotype mouse. We found significant interactions between gonadal sex and genotype for several behaviors. Finally, we asked if sibling pairs (as compared to non-siblings) would display qualitatively or quantitatively different behavior. In fact, XX females paired with a sibling were more social and less exploratory or investigative, whereas XY males exhibited less investigative and play soliciting behaviors in tests with siblings. Many neurobehavioral disorders, like autism spectrum disorder (ASD), are sexually dimorphic in incidence and patients interact less than normal with other children. Our results suggest that sex chromosome genes interact with gonadal hormones to shape the development of juvenile social behavior, and that social context can drastically alter sex differences. These data may have relevance for understanding the etiology of sexually dimorphic disorders such as ASD.

Of mice and rats: key species variations in the sexual differentiation of brain and behavior.

Mice and rats are important mammalian models in biomedical research. In contrast to other biomedical fields, work on sexual differentiation of brain and behavior has traditionally utilized comparative animal models. As mice are gaining in popularity, it is essential to acknowledge the differences between these two rodents. Here we review neural and behavioral sexual dimorphisms in rats and mice, which highlight species differences and experimental gaps in the literature, that are needed for direct species comparisons. Moving forward, investigators must answer fundamental questions about their chosen organism, and attend to both species and strain differences as they select the optimal animal models for their research questions.

Saccharomyces cerevisiae GATA sequences function as TATA elements during nitrogen catabolite repression and when Gln3p is excluded from the nucleus by overproduction of Ure2p.

Saccharomyces cerevisiae selectively uses good nitrogen sources (glutamine) in preference to poor ones (proline) by repressing GATA factor-dependent transcription of the genes needed to transport and catabolize poor nitrogen sources, a physiological process designated nitrogen catabolite repression (NCR). We show that some NCR-sensitive genes (CAN1, DAL5, DUR1,2, and DUR3) produce two transcripts of slightly different sizes. Synthesis of the shorter transcript is NCR-sensitive and that of the longer transcript is not. The longer transcript also predominates in gln3Delta mutants irrespective of the nitrogen source provided. We demonstrate that the longer mRNA species arises through the use of an alternative transcription start site generated by Gln3p-binding sites (GATAAs) being able to act as surrogate TATA elements. The ability of GATAAs to serve as surrogate TATAs, i.e. when synthesis of the shorter, NCR-sensitive transcripts are inhibited, correlates with sequestration of enhanced green fluorescent protein (EGFP)-Gln3p in the cytoplasm in a way that is indistinguishable from that seen with EGFP-Ure2p. However, when the shorter, NCR-sensitive DAL5 transcript predominates, EGFP-Gln3p is nuclear. These data suggest that the mechanism underlying NCR involves the cytoplasmic association of Ure2p with Gln3p, an interaction that prevents Gln3p from reaching it is binding sites upstream of NCR-sensitive genes.

Intracellular trafficking of variant chicken kidney AE1 anion exchangers: role Of alternative NH(2) termini in polarized sorting and Golgi recycling.

The variant chicken kidney AE1 anion exchangers differ only at the NH(2) terminus of their cytoplasmic domains. Transfection studies have indicated that the variant chicken AE1-4 anion exchanger accumulates in the basolateral membrane of polarized MDCK kidney epithelial cells, while the AE1-3 variant, which lacks the NH(2)-terminal 63 amino acids of AE1-4, primarily accumulates in the apical membrane. Mutagenesis studies have shown that the basolateral accumulation of AE1-4 is dependent upon two tyrosine residues at amino acids 44 and 47 of the polypeptide. Interestingly, either of these tyrosines is sufficient to direct efficient basolateral sorting of AE1-4. However, in the absence of both tyrosine residues, AE1-4 accumulates in the apical membrane of MDCK cells. Pulse-chase studies have shown that after delivery to the cell surface, newly synthesized AE1-4 is recycled to the Golgi where it acquires additional N-linked sugar modifications. This Golgi recycling activity is dependent upon the same cytoplasmic tyrosine residues that are required for the basolateral sorting of this variant transporter. Furthermore, mutants of AE1-4 that are defective in Golgi recycling are unable to associate with the detergent insoluble actin cytoskeleton and are rapidly turned over. These studies, which represent the first description of tyrosine-dependent cytoplasmic sorting signal for a type III membrane protein, have suggested a critical role for the actin cytoskeleton in regulating AE1 anion exchanger localization and stability in this epithelial cell type.

Genome-wide transcriptional analysis in S. cerevisiae by mini-array membrane hybridization.

Access to the powerful micro-array analytical methods used for genome-wide transcriptional analysis has so far been restricted by the high cost and/or lack of availability of the sophisticated instrumentation and materials needed to perform it. Mini-array membrane hybridization provides a less expensive alternative. The reliability of this technique, however, is not well documented and its reported use has, up to this point, been very limited. Our objective was to test whether or not mini-array membrane hybridization would reliably identify genes whose expression was controlled by a specific set of genetic and/or physiological signals. Our results demonstrate that mini-array hybridization can correctly identify genes whose expression is known to be controlled by the GATA-factor regulatory network in S. cerevisiae and in addition can reliably identify genes not previously reported to be associated with this nitrogen control system.

Chicken erythroid AE1 anion exchangers associate with the cytoskeleton during recycling to the Golgi.

Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some of the anion exchangers become detergent insoluble. The acquisition of detergent insolubility correlates with the association of the anion exchanger with cytoskeletal ankyrin. Reagents that inhibit different steps in the endocytic pathway, including 0.4 M sucrose, ammonium chloride, and brefeldin A, block the acquisition of endo F-resistant sugars and the acquisition of detergent insolubility by newly synthesized anion exchangers. The inhibitory effects of ammonium chloride on anion exchanger processing are rapidly reversible. Furthermore, AE1 anion exchangers become detergent insoluble more rapidly than they acquire endo F-resistant modifications in cells recovering from an ammonium chloride block. This suggests that the cytoskeletal association of the recycling anion exchangers occurs after release from the compartment where they accumulate due to ammonium chloride treatment, and prior to their transit through the Golgi. The recycling pool of newly synthesized anion exchangers is reflected in the steady-state distribution of the polypeptide. In addition to plasma membrane staining, anion exchanger antibodies stain a perinuclear compartment in erythroid cells. This perinuclear AE1-containing compartment is also stained by ankyrin antibodies and partially overlaps the membrane compartment stained by NBD C6-ceramide, a Golgi marker. Detergent extraction of erythroid cells in situ has suggested that a substantial fraction of the perinuclear pool of AE1 is cytoskeletal associated. The demonstration that erythroid anion exchangers interact with elements of the cytoskeleton during recycling to the Golgi suggests the cytoskeleton may be involved in the post-Golgi trafficking of this membrane transporter.

Fasting and postprandial lipid abnormalities in hypopituitary women receiving conventional replacement therapy.

Hypopituitary patients, particularly women, have excess mortality, mostly due to vascular disease. We have studied circulating lipid and lipoprotein concentrations, fasting and over 24 h, in hypopituitary women and men and in matched controls. Firstly, 67 hypopituitary patients (36 women) and 87 normal controls (54 women) were studied after an overnight fast. Secondly, 12 patients (6 women) and 14 matched controls (7 women) were studied over 24 h of normal meals and activity. The patients were all GH deficient and were replaced with cortisol, T4, and sex hormones where appropriate, but not with GH. In the first study, circulating triglycerides, total cholesterol, high density lipoprotein (HDL) cholesterol, and low density lipoprotein (LDL) cholesterol were measured after an overnight fast. In the second study, fasting levels of apolipoprotein B, apolipoprotein A1, and lipoprotein(a) were also measured, and then circulating triglyceride and total cholesterol concentrations were measured over 24 h. Fasting concentrations of triglyceride (mean +/- SEM, 1.73 +/- 0.22 vs. 1.11 +/- 0.09 mmol/L; P = 0.0025), total cholesterol (6.45 +/- 0.25 vs. 5.59 +/- 0.21 mmol/L; P = 0.002), LDL cholesterol (4.58 +/- 0.24 vs. 3.80 +/- 0.19 mmol/L; P = 0.007), and apolipoprotein B (135 +/- 10 vs. 111 +/- 9 mg/dL; P = 0.048) were elevated in hypopituitary compared to control women. The lipid alterations were observed in older and younger women and occurred independently of sex hormone or glucocorticoid replacement. Fasting values were not significantly different in hypopituitary and control men. Patients and controls (women and men) had similar fasting HDL cholesterol, apolipoprotein A1, and lipoprotein(a) concentrations. Although the differences that existed in fasting lipid values were most marked in women, the men were also abnormal in this respect, in that a higher proportion of hypopituitary than control men had total and LDL cholesterol above recommended values (> or = 6.2 and > or = 4.1 mmol/L, respectively). In the postprandial period (0730-2030 h), the areas under the curve (AUC) for circulating triglyceride and total cholesterol were significantly higher in hypopituitary than control women (P = 0.0089 and P = 0.0016, respectively). The AUC for triglyceride and total cholesterol over 24 h were also significantly increased (P = 0.009 and P = 0.0004, respectively). No significant differences were observed for postprandial and 24-h AUC for triglyceride and total cholesterol concentrations in men. We conclude that hypopituitarism with conventional replacement therapy is associated with unfavorable fasting and postprandial lipid and lipoprotein concentrations, particularly in women. The changes may contribute to the observed increased vascular morbidity and mortality.

Variant chicken kidney AE1 anion exchanger transcripts are derived from a single promoter by alternative splicing.

Previous studies have demonstrated that three variant transcripts, AE1-3, AE1-4 and AE1-5, are derived from the AE1 gene in chicken kidney. These variant transcripts encode AE1 anion exchangers that possess alternative N-terminal cytoplasmic domains. To determine the mechanisms involved in generating these transcripts, a genomic clone, containing the unique sequences at the 5' ends of the AE1-4 and AE1-5 transcripts, was isolated. Characterization of this clone revealed that the sequences at the 5' ends of the AE1-3, AE1-4 and AE1-5 transcripts were each present with an approx. 1.2-kb BamHI fragment of the chicken AE1 gene. RNA blotting and RNase protection analyses using probes derived from this genomic clone have shown that the AE1-4 variant corresponds to the approx. 4.5-kb chicken kidney AE1 transcript, while the AE1-5 variant corresponds to the approx. 5.1-kb transcript. These studies have shown that the AE1-5 transcript extends further 5' than had been previously shown from cDNA cloning studies, and contains the sequence present at the 5' end of the AE1-4 transcript. In addition, primer extension analyses have shown that the variant kidney AE1 transcripts initiate transcription from a common site. This result indicates that the expression of the AE1-3, AE1-4, and AE1-5 transcripts is regulated by a single promoter, P3, that is distinct from the P1 and P2 erythroid-specific promoters of the chicken AE1 gene.

Variant AE2 anion exchanger transcripts accumulate in multiple cell types in the chicken gastric epithelium.

Molecular analyses have resulted in the isolation of two chicken stomach AE2 anion exchanger cDNAs, AE2-1 and AE2-2. The approximately 4.3-kilobase (kb) AE2-1 cDNA contains an open reading frame that encodes a predicted polypeptide of approximately 135 kDa that is homologous to AE2 anion exchangers from other species. The partial approximately 1.7-kb AE2-2 cDNA, which differs from the AE2-1 cDNA in two regions, would be predicted to encode an AE2 polypeptide with an alternative N-terminal cytoplasmic tail. Examination of the distribution of these variant transcripts has revealed that AE2 transcripts ranging in size from approximately 4.4 to approximately 7.3 kb accumulate in various adult tissues. However, in the stomach, the unique sequence at the 5'-end of AE2-1 is preferentially associated with transcripts that range in size from approximately 4.5 to approximately 4.9 kb, while the unique sequence at the 5'-end of AE2-2 is preferentially associated with the approximately 7.3-kb AE2 RNA species. In situ hybridization analyses have further revealed that AE2 transcripts accumulate to very high levels within the acid-secreting epithelial cells of the profound gland in the stomach and, to a lesser extent, within the mucus-secreting cells of the superficial gland that line the stomach lumen. This result suggests that AE2 anion exchangers are involved in the regulation of intracellular pH in each of these gastric epithelial cell types.

Four variant chicken erythroid AE1 anion exchangers. Role of the alternative N-terminal sequences in intracellular targeting in transfected human erythroleukemia cells.

Four variant AE1 anion exchangers with predicted molecular masses of approximately 99, approximately 102, approximately 104, and approximately 108 kDa are expressed in chicken erythroid cells. These variant polypeptides differ in sequence only at the N terminus of their cytoplasmic domains. Molecular analyses have shown that transcripts derived from both of the erythroid-specific promoters, P1 and P2, encode all four of these AE1 anion exchanger variants. However, quantitative RNase protection analyses have shown that the transcripts derived from the P1 promoter are much more prevalent than those derived from the P2 promoter. Reverse transcriptase polymerase chain reaction studies have indicated that the extensive diversity in the transcripts derived from the AE1 gene occurs both in primitive and definitive lineage erythroid cells. Transient transfection analyses using human erythroleukemia cells have investigated the functional significance of the alternative sequences at the N terminus of these variant exchangers. These studies have shown that the erythroid AE1 variants are sorted to different membrane compartments in these cells. The approximately 99- and approximately 102-kDa variants are primarily sorted to the plasma membrane, whereas the approximately 108-kDa variant is retained in a perinuclear compartment. These results suggest that the alternative N-terminal cytoplasmic sequences of these polypeptides may serve as signals to direct these variant transporters to different membrane compartments within cells.

Variant chicken AE1 anion exchangers possess divergent NH(2)-terminal cytoplasmic domains.

Immunoblotting analyses have demonstrated that antibodies specific for the chicken erythroid AE1 anion exchanger recognize multiple polypeptides ranging in size from approximately 95 to 112 kDa in chicken kidney. To determine the origin of this diversity, we have cloned and characterized the kidney AE1 anion exchangers. These studies have shown that the kidney AE1 polypeptides are encoded by at least three transcripts, AE1-3, AE1-4, and AE1-5, which differ from the erythroid AE1-1 and AE1-2 transcripts in the sequences present at their 5'-ends. The AE1-3 and AE1-5 transcripts encode predicted polypeptides of approximately 94 kDa, which are identical to the erythroid AE1-1 anion exchanger except for the absence of the 78 NH(2)-terminal amino acids of the AE1-1 polypeptide. In contrast, the AE1-4 transcript encodes a predicted polypeptide of approximately 101 kDa, whose 21 NH(2)-terminal amino acids are unique. Characterization of the AE1 cDNAs has suggested that the AE1-3 and AE1-4 transcripts are generated by alternative splicing of a single primary transcript, while DNA blotting analyses have shown that the putative transcription initiation sites of the variant AE1-4 and AE1-5 transcripts lie several kilobases downstream of the transcription initiation sites of the erythroid AE1-1 and AE1-2 transcripts. These results suggest that the pattern of accumulation of the variant kidney AE1 anion exchangers is regulated by a complex pattern of alternative transcriptional initiation and differential RNA splicing.

Different Temporal and Spatial Gene Expression Patterns Occur during Anther Development.

We studied the temporal and spatial regulation of three mRNA sequence sets that are present exclusively, or at elevated levels, in the tobacco anther. One mRNA set accumulates in the tapetum and decays as the tapetum degenerates later in anther development. The second mRNA set accumulates after the tapetal-specific mRNAs, is localized within the stomium and connective, and also decays as these cell types degenerate during anther maturation. The third mRNA sequence set persists throughout anther development and is localized within most anther tissues. A tapetal-specific gene, designated as TA29, was isolated from a tobacco genome library. Runoff transcription studies and experiments with chimeric [beta]-glucuronidase and diphtheria toxin A-chain genes showed that the TA29 gene is regulated primarily at the transcriptional level and that a 122-base pair 5[prime] region can program the tapetal-specific expression pattern. Destruction of the tapetum by the cytotoxic gene had no effect on the differentiation and/or function of surrounding sporophytic tissues but led to the production of male-sterile plants. Together, our studies show that several independent gene expression programs occur during anther development and that these programs correlate with the differentiated state of specific anther cell types.

Cell lineage-specific programs of expression of multiple actin genes during sea urchin embryogenesis.

We have determined spatial patterns of expression of individual actin genes in embryos of the sea urchin Strongylocentrotus purpuratus. Radioactively labeled probes specific for each of five cytoplasmic-type (Cy) and the single muscle-type (M) mRNAs were hybridized in situ to sections of fixed embryos. M actin mRNA appears only late in development and is confined to a few cells associated with the coelomic rudiments. The five Cy mRNAs fall into three sets, whose times and sites of expression during development are highly distinctive. Different cell lineages express messages of one or more of these sets, but never all three. Although all Cy actin mRNAs exhibit monophasic accumulation in the RNA of whole embryos during the course of development, such accumulation in many cases results from the summation of both increases and decreases in abundance within individual sets of cells. Within the genomic linkage group CyI-CyIIa-CyIIb, expression of CyI and CyIIb appears to be co-ordinate, and quite distinct from that of CyIIa. CyI and CyIIb are expressed in all lineages at some point in embryogenesis, but confined mainly to oral ectoderm and portions of the gut of the pluteus larva. CyIIa mRNAs are restricted to mesenchyme lineages throughout late gastrula stage, and subsequently accumulate in parts of the gut. The CyIIIa and CyIIIb genes, which form a separate linkage group, are expressed only in aboral ectoderm and its precursors. Furthermore, CyIII messages are the only detectable actin mRNAs in this cell lineage after late blastula stage.