Oxytocin is required for nursing but is not essential for parturition or reproductive behavior.

Oxytocin, a neurohypophyseal hormone, has been traditionally considered essential for mammalian reproduction. In addition to uterine contractions during labor and milk ejection during nursing, oxytocin has been implicated in anterior pituitary function, paracrine effects in the testis and ovary and the neural control of maternal and sexual behaviors. To determine the essential role(s) of oxytocin in mammalian reproductive function, mice deficient in oxytocin have been generated using embryonic stem cell technology. A deletion of exon 1 encoding the oxytocin peptide was generated in embryonic stem cells at a high frequency and was successfully transmitted in the germ line. Southern blot analysis of genomic DNA from homozygote offspring and in situ hybridization with an exonic probe 3' of the deletion failed to detect any oxytocin or neurophysin sequences, respectively, confirming that the mutation was a null mutation. Mice lacking oxytocin are both viable and fertile. Males do not have any reproductive behavioral or functional defects in the absence of oxytocin. Similarly, females lacking oxytocin have no obvious deficits in fertility or reproduction, including gestation and parturition. However, although oxytocin-deficient females demonstrate normal maternal behavior, all offspring die shortly after birth because of the dam's inability to nurse. Postpartum injections of oxytocin to the oxytocin deficient mothers restore milk ejection and rescue the offspring. Thus, despite the multiple reproductive activities that have been attributed to oxytocin, oxytocin plays an essential role only in milk ejection in the mouse.

Activation domain-mediated enhancement of activator binding to chromatin in mammalian cells.

DNA binding by transcriptional activators is typically an obligatory step in the activation of gene expression. Activator binding and subsequent steps in transcription are repressed by genomic chromatin. Studies in vitro have suggested that overcoming this repression is an important function of some activation domains. Here we provide quantitative in vivo evidence that the activation domain of GAL4-VP16 can increase the affinity of GAL4 for its binding site on genomic DNA in mammalian cells. Moreover, the VP16 activation domain has a much greater stimulatory effect on expression from a genomic reporter gene than on a transiently transfected reporter gene, where factor binding is more permissive. We found that not all activation domains showed a greater activation potential in a genomic context, suggesting that only some activation domains can function in vivo to alleviate the repressive effects of chromatin. These data demonstrate the importance of activation domains in relieving chromatin-mediated repression in vivo and suggest that one way they function is to increase binding of the activator itself.

The human AQP4 gene: definition of the locus encoding two water channel polypeptides in brain.

The aquaporin family of membrane water transport proteins are expressed in diverse tissues, and in brain the predominant water channel protein is AQP4. Here we report the isolation and characterization of the human AQP4 cDNAs and genomic DNA. Two cDNAs were isolated corresponding to the two initiating methionines (M1 in a 323-aa polypeptide and M23 in a 301-aa polypeptide) previously identified in rat [Jung, J.S., Bhat, R.V., Preston, G.M., Guggino, W.B. & Agre, P. (1994) Proc. Natl. Acad. Sci. USA 91, 13052-13056]. Similar to other aquaporins, the AQP4 gene is composed of four exons encoding 127, 55, 27, and 92 amino acids separated by introns of 0.8, 0.3, and 5.2 kb. Unlike other aquaporins, an alternative coding initiation sequence (designated exon 0) was located 2.7 kb upstream of exon 1. When spliced together, M1 and the subsequent 10 amino acids are encoded by exon 0; the next 11 amino acids and M23 are encoded by exon 1. Transcription initiation sites have been mapped in the proximal promoters of exons 0 and 1. RNase protection revealed distinct transcripts corresponding to M1 and M23 mRNAs, and AQP4 immunoblots of cerebellum demonstrated reactive polypeptides of 31 and 34 kDa. Using a P1 and a lambda EMBL subclone, the chromosomal site of the human AQP4 gene was mapped to chromosome 18 at the junction of q11.2 and q12.1 by fluorescence in situ hybridization. These studies may now permit molecular characterization of AQP4 during human development and in clinical disorders.

Organization and chromosomal localization of the gene encoding the mouse acid labile subunit of the insulin-like growth factor binding complex.

After birth, most of insulin-like growth factor I and II (IGFs) circulate as a ternary complex formed by the association of IGF binding protein 3-IGF complexes with a serum protein called acid-labile subunit (ALS). ALS retains the IGF binding protein-3-IGF complexes in the vascular compartment and extends the t1/2 of IGFs in the circulation. Synthesis of ALS occurs mainly in liver after birth and is stimulated by growth hormone. To study the basis for this regulation, we cloned and characterized the mouse ALS gene. Comparison of genomic and cDNA sequences indicated that the gene is composed of two exons separated by a 1126-bp intron. Exon 1 encodes the first 5 amino acids of the signal peptide and contributes the first nucleotide of codon 6. Exon 2 contributes the last 2 nt of codon 6 and encodes the remaining 17 amino acids of the signal peptide as well as the 580 amino acids of the mature protein. The polyadenylylation signal, ATTAAA, is located 241 bp from the termination codon. The cDNA and genomic DNA diverge 16 bp downstream from this signal. Transcription initiation was mapped to 11 sites over a 140-bp TATA-less region. The DNA fragment extending from nt -805 to -11 (ATG, +1) directed basal and growth hormone-regulated expression of a luciferase reporter plasmid in the rat liver cell line H4-II-E. Finally, the ALS gene was mapped to mouse chromosome 17 by fluorescence in situ hybridization.

Sequence and expression of the murine Hoxd-3 homeobox gene.

Murine Hoxd-3 (Hox 4.1) genomic DNA and cDNA and Hoxa-3 (Hox 1.5) cDNA were cloned and sequenced. The homeodomains of Hoxd-3 and Hoxa-3 and regions before and after the homeodomain are highly conserved. Both Hoxa-3 and Hoxa-3 proteins have a proline-rich region that contains consensus amino acid sequences for binding to Src homology 3 domains of some signal transduction proteins. Northern blot analysis of RNA from 8- to 11-day-old mouse embryos revealed a 4.3-kb species of Hoxd-3 RNA, whereas a less abundant 3.0-kb species of Hoxd-3 RNA was found in RNA from 9- to 11-day-old embryos. Two species of Hoxd-3 poly(A)+ RNA, 4.3 and 6.0 kb in length, were found in poly(A)+ RNA from adult mouse kidney, but not in RNA from other adult tissues tested. Hoxd-3 mRNA was detected by in situ hybridization in 12-, 14-, and 17-day-old mouse embryos in the posterior half of the myelencephalon, spinal cord, dorsal root ganglia, first cervical vertebra, thyroid gland, kidney tubules, esophagus, stomach, and intestines.

An archaebacterial homologue of the essential eubacterial cell division protein FtsZ.

Life falls into three fundamental domains--Archaea, Bacteria, and Eucarya (formerly archaebacteria, eubacteria, and eukaryotes,. respectively). Though Archaea lack nuclei and share many morphological features with Bacteria, molecular analyses, principally of the transcription and translation machineries, have suggested that Archaea are more related to Eucarya than to Bacteria. Currently, little is known about the archaeal cell division apparatus. In Bacteria, a crucial component of the cell division machinery is FtsZ, a GTPase that localizes to a ring at the site of septation. Interestingly, FtsZ is distantly related in sequence to eukaryotic tubulins, which also interact with GTP and are components of the eukaryotic cell cytoskeleton. By screening for the ability to bind radiolabeled nucleotides, we have identified a protein of the hyperthermophilic archaeon Pyrococcus woesei that interacts tightly and specifically with GTP. Furthermore, through screening an expression library of P. woesei genomic DNA, we have cloned the gene encoding this protein. Sequence comparisons reveal that the P. woesei GTP-binding protein is strikingly related in sequence to eubacterial FtsZ and is marginally more similar to eukaryotic tubulins than are bacterial FtsZ proteins. Phylogenetic analyses reinforce the notion that there is an evolutionary linkage between FtsZ and tubulins. These findings suggest that the archaeal cell division apparatus may be fundamentally similar to that of Bacteria and lead us to consider the evolutionary relationships between Archaea, Bacteria, and Eucarya.

Two genes abrogate the inhibition of murine hepatocarcinogenesis by ovarian hormones.

Hormonal and genetic factors strongly influence the susceptibility of inbred mice to hepatocarcinogenesis. Female C57BR/cdJ (BR) mice are extremely susceptible to liver tumor induction relative to other strains because they are genetically insensitive to the inhibition of hepatocarcinogenesis by ovarian hormones. To determine the genetic basis for the sensitivity of BR mice relative to resistant C57BL/6J (B6) mice, we treated 12-day-old B6BRF1 x B6 and B6BRF1 x B6BRF1 (F2) animals with N,N-diethylnitrosamine (0.1 micromol/g of body weight) and enumerated liver tumors at 32 weeks of age in males and at 50 weeks in females. Genomic DNA samples from backcross and F2 mice were analyzed for 70 informative simple sequence length polymorphism markers. Genetic markers on chromosome 17 (D17Mit21) and chromosome 1 (D1Mit33) cosegregated with high tumor multiplicity in both sexes. Together, these loci [designated Hcf1 and Hcf2 (Hepatocarcinogenesis in females), respectively] account for virtually all of the difference in sensitivity between BR and B6 mice. The Hcf1 locus accounts for a majority of the higher susceptibility of BR mice of both sexes. Backcross female mice heterozygous at both loci (33 +/- 23 tumors per mouse) and at Hcf1 only (17 +/- 18) were 15- and 8-fold more sensitive, respectively, than mice homozygous for the B6 alleles at Hcf1 and Hcf2 (2.2 +/- 3.9). In backcross male mice, the double heterozygotes (35 +/- 22) and Hcf1 heterozygotes (28 +/- 12) were 5.4- and 4.3-fold more sensitive than mice homozygous for B6 alleles at both loci (6.5 +/- 5.4).

The adipocyte specific transcription factor C/EBPalpha modulates human ob gene expression.

The ob gene product, leptin, apparently exclusively expressed in adipose tissue, is a signaling factor regulating body weight homeostasis and energy balance. ob gene expression is increased in obese rodents and regulated by feeding, insulin, and glucocorticoids, which supports the concept that ob gene expression is under hormonal control, which is expected for a key factor controlling body weight homeostasis and energy balance. In humans, ob mRNA expression is increased in gross obesity; however, the effects of the above factors on human ob expression are unknown. We describe the structure of the human ob gene and initial functional analysis of its promoter. The human ob gene's three exons cover approximately 15 kb of genomic DNA. The entire coding region is contained in exons 2 and 3, which are separated by a 2-kb intron. The first small 30-bp untranslated exon is located >10.5 kb upstream of the initiator ATG codon. Three kilobases of DNA upstream of the transcription start site has been cloned and characterized. Only 217 bp of 5' sequence are required for basal adipose tissue-specific expression of the ob gene as well as enhanced expression by C/EBPalpha. Mutation of the single C/EBPalpha site in this region abolished inducibility of the promoter by C/EBPalpha in cotransfection assays. The gene structure will facilitate our analysis of ob mutations in human obesity, whereas knowledge of sequence elements and factors regulating ob gene expression should be of major importance in the prevention and treatment of obesity.

Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility.

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis. To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2 -/-) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile. However, neither meiosis nor fertility was affected in female Hsp70-2 -/- mice. We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2 -/- spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions. Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2 -/- mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis.

Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals.

The jaagsiekte sheep retrovirus (JSRV), which appears to be a type B/D retrovirus chimera, has been incriminated as the cause of ovine pulmonary carcinoma. Recent studies suggest that the sequences related to this virus are found in the genomes of normal sheep and goats. To learn whether there are breeds of sheep that lack the endogenous viral sequences and to study their distribution among other groups of mammals, we surveyed several domestic sheep and goat breeds, other ungulates, and various mammal groups for sequences related to JSRV. Probes prepared from the envelope (SU) region of JSRV and the capsid (CA) region of a Peruvian type D virus related to JSRV were used in Southern blot hybridization with genomic DNA followed by low- and high-stringency washes. Fifteen to 20 CA and SU bands were found in all members of the 13 breeds of domestic sheep and 6 breeds of goats tested. There were similar findings in 6 wild Ovis and Capra genera. Within 22 other genera of Bovidae including domestic cattle, and 7 other families of Artiodactyla including Cervidae, there were usually a few CA or SU bands at low stringency and rare bands at high stringency. Among 16 phylogenetically distant genera, there were generally fewer bands hybridizing with either probe. These results reveal wide-spread phylogenetic distribution of endogenous type B and type D retroviral sequences related to JSRV among mammals and argue for further investigation of their potential role in disease.

Molecular characterization of a positively photoregulated nuclear gene for a chloroplast RNA polymerase sigma factor in Cyanidium caldarium.

We have cloned the gene for a putative chloroplast RNA polymerase sigma factor from the unicellular rhodophyte Cyanidium caldarium. This gene contains an open reading frame encoding a protein of 609 amino acids with domains highly homologous to all four conserved regions found in bacterial and cyanobacterial sigma 70-type subunits. When Southern blots of genomic DNA were hybridized to the "rpoD box" oligonucleotide probe, up to six hybridizing hands were observed. Transcripts of the sigma factor gene were undetectable in RNA from dark-grown cells but were abundant in the poly(A)+ fraction of RNA from illuminated cells. The sigma factor gene was expressed in Escherichia coli, and antibodies against the expressed sigma factor fusion protein cross-reacted with a 55-kDa protein in partially purified chloroplast RNA polymerase. Antibodies directed against a cyanobacterial RNA polymerase sigma factor also cross-reacted with a 55-kDa protein in the same enzyme preparation. Immunoprecipitation experiments showed that this enzyme preparation contains proteins with the same molecular weights as the alpha, beta, beta', and beta" subunits of chloroplast RNA polymerase in higher plants. This study identifies a gene for a plastid RNA polymerase sigma factor and indicates that there may be a family of nuclear-encoded sigma factors that recognize promoters in subsets of plastid genes and regulate differential gene expression at the transcriptional level.

A high-resolution physical map of human chromosome 11.

The development of a highly reliable physical map with landmark sites spaced an average of 100 kbp apart has been a central goal of the Human Genome Project. We have approached the physical mapping of human chromosome 11 with this goal as a primary target. We have focused on strategies that would utilize yeast artificial chromosome (YAC) technology, thus permitting long-range coverage of hundreds of kilobases of genomic DNA, yet we sought to minimize the ambiguities inherent in the use of this technology, particularly the occurrence of chimeric genomic DNA clones. This was achieved through the development of a chromosome 11-specific YAC library from a human somatic cell hybrid line that has retained chromosome 11 as its sole human component.To maximize the efficiency of YAC contig assembly and extension, we have employed an Alu-PCR-based hybridization screening system. This system eliminates many of the more costly and time-consuming steps associated with sequence tagged site content mapping such as sequencing, primer production, and hierarchical screening, resulting in greater efficiency with increased throughput and reduced cost. Using these approaches, we have achieved YAC coverage for >90% of human chromosome 11, with an average intermarker distance of <100 kbp. Cytogenetic localization has been determined for each contig by fluorescent in situ hybridization and/or sequence tagged site content. The YAC contigs that we have generated should provide a robust framework to move forward to sequence-ready templates for the sequencing efforts of the Human Genome Project as well as more focused positional cloning on chromosome 11.

Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice.

Gene transduction of pluripotent human hematopoietic stem cells (HSCs) is necessary for successful gene therapy of genetic disorders involving hematolymphoid cells. Evidence for transduction of pluripotent HSCs can be deduced from the demonstration of a retroviral vector integrated into the same cellular chromosomal DNA site in myeloid and lymphoid cells descended from a common HSC precursor. CD34+ progenitors from human bone marrow and mobilized peripheral blood were transduced by retroviral vectors and used for long-term engraftment in immune-deficient (beige/nude/XIS) mice. Human lymphoid and myeloid populations were recovered from the marrow of the mice after 7-11 months, and individual human granulocyte-macrophage and T-cell clones were isolated and expanded ex vivo. Inverse PCR from the retroviral long terminal repeat into the flanking genomic DNA was performed on each sorted cell population. The recovered cellular DNA segments that flanked proviral integrants were sequenced to confirm identity. Three mice were found (of 24 informative mice) to contain human lymphoid and myeloid populations with identical proviral integration sites, confirming that pluripotent human HSCs had been transduced.

Cloning and expression of a gene encoding an integrin-like protein in Candida albicans.

The existence of integrin-like proteins in Candida albicans has been postulated because monoclonal antibodies to the leukocyte integrins alpha M and alpha X bind to blastospores and germ tubes, recognize a candidal surface protein of approximately 185 kDa, and inhibit candidal adhesion to human epithelium. The gene alpha INT1 was isolated from a library of C. albicans genomic DNA by screening with a cDNA probe from the transmembrane domain of human alpha M. The predicted polypeptide (alpha Int1p) of 188 kDa contains several motifs common to alpha M and alpha X: a putative I domain, two EF-hand divalent cation-binding sites, a transmembrane domain, and a cytoplasmic tail with a single tyrosine residue. An internal RGD tripeptide is also present. Binding of anti-peptide antibodies raised to potential extracellular domains of alpha Int1p confirms surface localization in C. albicans blastopores. By Southern blotting, alpha INT1 is unique to C. albicans. Expression of alpha INT1 under control of a galactose-inducible promoter led to the production of germ tubes in haploid Saccharomyces cerevisiae and in the corresponding ste12 mutant. Germ tubes were not observed in haploid yeast transformed with vector alone, in transformants expressing a galactose-inducible gene from Chlamydomonas, or in transformants grown in the presence of glucose or raffinose. Transformants producing alpha Int1p bound an anti-alpha M monoclonal antibody and exhibited enhanced aggregation. Studies of alpha Int1p reveal novel roles for primitive integrin-like proteins in adhesion and in STE12-independent morphogenesis.

Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes.

A family of interferon (IFN) regulatory factors (IRFs) have been shown to play a role in transcription of IFN genes as well as IFN-stimulated genes. We report the identification of a member of the IRF family which we have named IRF-3. The IRF-3 gene is present in a single copy in human genomic DNA. It is expressed constitutively in a variety of tissues and no increase in the relative steady-state levels of IRF-3 mRNA was observed in virus-infected or IFN-treated cells. The IRF-3 gene encodes a 50-kDa protein that binds specifically to the IFN-stimulated response element (ISRE) but not to the IRF-1 binding site PRD-I. Overexpression of IRF-3 stimulates expression of the IFN-stimulated gene 15 (ISG15) promoter, an ISRE-containing promoter. The murine IFNA4 promoter, which can be induced by IRF-1 or viral infection, is not induced by IRF-3. Expression of IRF-3 as a Gal4 fusion protein does not activate expression of a chloramphenicol acetyltransferase reporter gene containing repeats of the Gal4 binding sites, indicating that this protein does not contain the transcription transactivation domain. The high amino acid homology between IRF-3 and ISG factor 3 gamma polypeptide (ISGF3 gamma) and their similar binding properties indicate that, like ISGF3 gamma, IRF-3 may activate transcription by complex formation with other transcriptional factors, possibly members of the Stat family. Identification of this ISRE-binding protein may help us to understand the specificity in the various Stat pathways.