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Expression of the proto-oncogene int-1 is restricted to postmeiotic male germ cells and the neural tube of mid-gestational embryos
Abstract
The int-1 proto-oncogene is transcriptionally activated in mammary tumors by mouse mammary tumor virus insertion mutations and is normally expressed only in adult mouse testes and mid-gestational embryos. We have used anatomical dissection of embryos, germ-cell fractionation, peripuberal expression studies, and spermatogenesis mutants to identify more precisely the tissues and cells that contain int-1 RNA. In the testis, int-1 RNA is detected only in postmeiotic germ cells undergoing differentiation from round spermatids into mature spermatozoa. In embryos 11-15 days after conception, expression of the gene is restricted to the developing central nervous system in regions of the neural tube other than the telencephalon. Our findings suggest that int-1 mediates developmental events at these two sites.
... It had been previously observed that some genes selectively expressed in testis also displayed elevated levels of mRNA in brain (Kilpatrick and Millette, 1986;Gizang-Ginsberg and Wolgemuth, 1987;Shackleford and Varmus, 1987; F ig u re 4.7 Emoult -Lange et al., 1989) and, to a lesser extent, in colon . A further northern blot experiment was therefore performed to evaluate whether Zfp-35 was expressed to a significant degree in these tissues. 2 8 S 1 8 S mature into resting spermatocytes at around 12 days after birth (Bellve, 1979). ...
... The data in Figure 4.10 suggest that Tcp-1, a member of the t-complex (Willison et al., 1986) and whose gene product is a component of the trans-golgi network (Willison et al., 1989), is also upregulated at the pachytene stage of prophase. The proto-oncogenes int-1 and c-abl both exhibit spermatid-specific transcription with int-1 appearing in both round and elongating spermatids (Shackleford and Varmus, 1987) whilst a novel c-abl transcript is limited to the elongating spermatids (Ponzetto and Wolgemuth, 1985). These haploid-specific events occur after the high level of Zfp-35 RNA has begun to decline. ...
The zinc finger domain is a sequence-specific DNA binding motif that was originally identified in the Xenopus laevis transcription factor IIIA (TFIIIA), and subsequently observed in the Kruppel segmentation gene of Drosophila melanogaster and the yeast transcription factor ADR1. Such extensive phylogenetic conservation of zinc finger structures in these regulatory genes suggested that genes containing them might also serve important regulatory functions during mammalian development. In this study an originalities encoding a sequence conserved between TFIIIA, Kruppel, and ADR1 was used to screen mammalian cDNA libraries with the objective of identifying zinc finger genes selectively expressed during human B-lymphocyte and mouse germ cell development. A large number of cDNA clones were isolated from a human B-lymphocyte library. The sequence of a 1.8kb cDNA, ZFP36-1.8, revealed that its corresponding gene contained a minimum of 14 contiguous zinc fingers and an N-terminal non-finger region. At high stringency of hybridisation in Southern analysis this cDNA identified a large family of 20-30 highly related genomic DNA EcoRI fragments. Pulsed field gel analysis suggested that these fragments were clustered in the genome. The subfamily of sequences defined by ZFP36-1.8 was differentially transcribed in a wide range of cell types, although the transcription patterns in Northern analyses did partially overlap. Zinc finger cDNA clones were also isolated from an 11.5 day post coitum (p.c.) mouse urogenital ridge library. One was derived from a 2.4kb mRNA encoding a protein with a block of 18 zinc finger domains and an N-terminal region of 79 amino acids rich in acidic residues. Such acidic domains are a characteristic feature of the activation domains of many transcription factors. The corresponding gene, Zfp-35, was shown to be a single copy gene at high stringency of hybridisation. The 2.4kb mRNA was selectively expressed in adult testis by comparison with embryonic gonads. The expression of this mRNA was analysed in a variety of adult tissues, whole testes of prepuberal adult XY mice, and germ cells isolated at different stages of development from XY testes. Combined with in situ hybridisation to mRNA in sections from adult XY testes, these studies showed that a large increase in Zfp-35 expression was restricted to spermatocytes at the pachytene stage of meiotic prophase. Cosmid clones covering the Zfp-35 gene were characterised. The first exon of the 2.4kb transcription unit contained 53 nucleotides of the 5' untranslated region, exon 2 contained the remaining 5' untranslated region plus the first 17 codons of the open reading frame, and the third exon corresponded to the remainder of the mRNA including the acidic and 18 zinc finger domains of the open reading frame. The cognate gene could be identified in rat, human, whale, and horse genomic DNA. In situ hybridisation to mouse metaphase chromosomes revealed that Zfp-35 localised to a region of chromosome 18 encompassing bands B3 to C.
... PC12 is a rat pheochromocytoma cell line of neural crest lineage (Greene and Tischler, 1976). The neural crest and its derivatives arise from the Wnt-1-expressing dorsal region of the midbrain after closure of the neural tube (Shackleford and Varmus, 1987). Expression of Wnt-1 induces distinct morphological and biochemical changes in PC12 cells, including conversion from a rounded to a flattened, epithelial-like morphology, altered expression of E-cadherin, -catenin and plakoglobin, and increased calcium-dependent cell-cell adhesion (Bradley et al., 1993;Shackleford et al., 1993;Giarre et al., 1998). ...
... During the development of the mammalian nervous system, the cells that form the neural crest arise from the dorsal midline region after closure of the neural tube (Anderson, 1993). The expression of Wnt-1 in migrating neural crest cells (Echelard et al., 1994) originates from a Wnt-1 expressing region of the dorsal CNS (Shackleford et al., 1987;Wilkinson et al., 1987). Wnt-1 expression is critical for cell sorting, diversification and for the development of neural crest derivatives (Ikeya et al., 1997;Dorsky et al., 1998). ...
... Individuals with homozygous WNT1 mutations manifest severe skeletal abnormalities, reduced bone density and multiple fractures, growth delay and typical dominant OI extraskeletal findings such as blue sclerae and dentinogenesis imperfecta [114]. Developmental defects in the central nervous system (CNS) associated with intellectual disability are also occasionally described and may be linked to WNT1 expression in brain tissue [115,116]. WNT1 mutations Fig. 3. TRIC-B, SP2, OASIS and SPARC function in bone homeostasis. Collagen is synthesized within the ER, the major intracellular Ca 2+ store. ...
The limited accessibility of bone and its mineralized nature have restricted deep investigation of its biology. Recent breakthroughs in identification of mutant proteins affecting bone tissue homeostasis in rare skeletal diseases have revealed novel pathways involved in skeletal development and maintenance. The characterization of new dominant, recessive and X‐linked forms of the rare brittle bone disease osteogenesis imperfecta (OI) and other OI‐related bone fragility disorders was a key player in this advance. The development of in vitro models for these diseases along with the generation and characterization of murine and zebrafish models contributed to dissecting previously unknown pathways. Here, we describe the most recent advances in the understanding of processes involved in abnormal bone mineralization, collagen processing and osteoblast function, as illustrated by the characterization of new causative genes for OI and OI‐related fragility syndromes. The coordinated role of the integral membrane protein BRIL and of the secreted protein PEDF in modulating bone mineralization as well as the function and cross‐talk of the collagen‐specific chaperones HSP47 and FKBP65 in collagen processing and secretion are discussed. We address the significance of WNT ligand, the importance of maintaining endoplasmic reticulum membrane potential and of regulating intramembrane proteolysis in osteoblast homeostasis. Moreover, we also examine the relevance of the cytoskeletal protein plastin‐3 and of the nucleotidyltransferase FAM46A. Thanks to these advances, new targets for the development of novel therapies for currently incurable rare bone diseases have been and, likely, will be identified, supporting the important role of basic science for translational approaches.
... The Drosophila Wnt-1 homologue is the wingEess (wg) gene, a well-Characterized member of the segment polarity class of genes and essential for normal development of the fruit fly (Morata and Lawrence, 1977;Rijsewijk et al., 1987). Furthermore, in mice, expression of the Wnt-1 is restricted develop-mentally to testes of adults and to dorsal midline cells in the developing neural tube (Jakobovits et al., 1986;Shackelford and Varmus, 1987;Wilkinson et al., 1987). Based on this restricted pattern of expression, it had been postulated that the Wnt-1 gene could be an essential component in nervous system development. ...
... Recently, several vertebrate genes have been isolated that have DNA sequences related to the genes in Drosophila that regulate development. For instance, the murine oncogene int-l, which is related to the Drosophila gene wingless, is expressed in a subset of cells at the lateral edge of the neural plate (Shackelford & Varmus, 1987;Wilkinson, Bailes, & McMahon, 1987). Also, the POU-domain genes encode a family of proteins that are expressed in various subsets of cells in the developing and the adult mammalian nervous system (He, Treacy, Simmons, Ingraham, Swanson, & Rosenfeld, 1989). ...
Developmental disorders affecting intelligence and behavior are among the most mystifying diseases of humankind. This is because they are primarily caused by abnormalities in the development of the most complex human organ, the brain. Perhaps the reason so little is understood about mental retardation, autism, childhood schizophrenia, attention deficit disorder, specific learning disorders, and Tourette disorder is that very little is still understood about how a normal brain develops. The premise of this chapter is that the key to understand these illnesses is an understanding of the processes involved in neurodevelopment.
... Thus, the expression of DGS-G in adult testis does not preclude its playing a role in embryonic development. Further examples include, Hoxa-4 and int-1 which are expressed in the central nervous system of developing embryos, but demonstrate adult expression exclusively in testis (37)(38)(39). Given the central role of protein kinases in coordinating the eukaryotic cell's response to external and internal signals, DGS-G is an appealing candidate for DGS/VCFS. ...
The majority of patients with DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS) have a microdeletion of 22q11. Using translocation breakpoints and fluorescence in situ hybridization analysis (FISH), the minimal DiGeorge critical region (MDGCR) has been narrowed to 250 kb in the vicinity of D22S75 (N25). The construction of a detailed transcription map covering the MDGCR is an essential first step toward the identification of genes important to the etiology of DGSNCFS, two complex disorders. We have identified a minimum of 11 transcription units encoded in the MDGCR using a combination of methods including cDNA selection, RT-PCR, RACE and genomic sequencing. This approach is somewhat unique and may serve as a model for gene identification. Of the 11 transcripts, one is the previously reported DGCR2/IDD/LAN gene, and three revealed a high level of similarity to mammalian genes : a Mus musculus serine/threonine kinase, a rat tricarboxylate transport protein and a bovine clathrin heavy chain. The remaining transcripts do not demonstrate any significant homology to genes of known function. The identification of these transcription units in the MDGCR will facilitate their further characterization and help elucidate their role in the etiology of DGSNCFS.
... Although the detection of HLA molecules on the surface of spermatozoa is at odds with our present knowledge of the function of these antigens and the maturation process of spermatozoa, it should not be regarded as a mere curiosity. Other genes that do not have an obvious role in spermiogenesis, such as oncogenes [46] and HSP-70 [47], are known to be postmeiotically transcribed. This reveals our lack of knowledge on the function of these genes in normal development. ...
A diploid expression of class I and class II human leukocyte antigens (HLA) has been found in purified spermatozoa by using double fluorescence labeling cytofluorometry and relevant monoclonal antibodies; this expression has been confirmed for the first time by the analysis of specific HLA mRNA and metabolic 35 S labeling followed by immunoprecipitation, which demonstrates an active ongoing translation of HLA proteins in ger-minal cells. Long-living mRNA coming from diploid germinal cells may be translated to HLA molecules in spermatozoa. This translation is controlled (or at least inversely correlated) by a testicular hormone (inhibin) in a cyclic fashion. Remarkably, serum levels of inhibin, synthesized by Leydig and Sertoli cells, follow a 12-to 13-day cycle, with a peak level at Day 6; this is probably controlled by FSH (not cyclic in males) and other tes-ticular and/or unknown hormones. Peak levels of inhibin concur with the lower density and percentage of spermatozoa expressing both HLA class I and II molecules (close to 3% by cytofluo-rometry); lowest levels of inhibin coincide with the highest numbers (35-40%) of spermatozoa positive for both HLA molecules and a higher surface density. These observations could put to an end a disconcerting and long-lasting controversy on the expres-sion/non-expression of HLA antigens on spermatozoa. The possibility that HLA-bearing spermatozoa are more capacitated for fertilization than those that do not bear HLA, and the implications of our results on male fertility control are also discussed.
... However, these mechanisms do not appear to explain the marked decrease in STRA6 in adipose tissue of high-fat-diet-fed mice or ob/ob mice since retinoic acid levels and retinoic acid receptor signaling are not reduced in adipose tissue in these models (61; data not shown). Furthermore, p53 is increased in ob/ob mouse adipose tissue (62), and Wnt-1 is normally restricted to the testes in adult mice (63,64). In future studies, it would be of interest to determine if other Wnt proteins regulate STRA6 expression in adipose tissue. ...
To investigate the mechanisms by which elevated retinol-binding protein 4 (RBP4) causes insulin resistance, we studied the
role of the high-affinity receptor for RBP4, STRA6 (stimulated by retinoic acid), in insulin resistance and obesity. In high-fat-diet-fed
and ob/ob mice, STRA6 expression was decreased 70 to 95% in perigonadal adipocytes and both perigonadal and subcutaneous adipose stromovascular
cells. To determine whether downregulation of STRA6 in adipocytes contributes to insulin resistance, we generated adipose-Stra6−/− mice. Adipose-Stra6−/− mice fed chow had decreased body weight, fat mass, leptin levels, insulin levels, and adipocyte number and increased expression
of brown fat-selective markers in white adipose tissue. When fed a high-fat diet, these mice had a mild improvement in insulin
sensitivity at an age when adiposity was unchanged. STRA6 has been implicated in retinol uptake, but retinol uptake and the
expression of retinoid homeostatic genes (encoding retinoic acid receptor β [RARβ], CYP26A1, and lecithin retinol acyltransferase)
were not altered in adipocytes from adipose-Stra6−/− mice, indicating that retinoid homeostasis was maintained with STRA6 knockdown. Thus, STRA6 reduction in adipocytes in adipose-Stra6−/− mice fed chow resulted in leanness, which may contribute to their increased insulin sensitivity. However, in wild-type mice
with high-fat-diet-induced obesity and in ob/ob mice, the marked downregulation of STRA6 in adipocytes and adipose stromovascular cells does not compensate for obesity-associated
insulin resistance.
... VOL. 11, 1991 (17,21,36,40). Effects of HSV-1 TK expression on spermatogenesis. ...
Wereported previously that theherpes simplex virus type1(HSV-1) thymidine kinase reporter gene(tk) was expressed inthetestes oftransgenic micewhencoupled tothepromoter ofaliver-specific mouse majorurinary protein (MUP)gene.Herewe showthatHSV-1tkisalsoexpressed inthetestis whencoupled toa MUP pseudogene promoter, toatruncated MUP promoter thatisnotactive intheliver, andtothepromoterofthe bovine thyroglobulin gene.Furthermore, HSV-1tkitself was expressed inthetestis, although itsnormal expression hadbeendisabled byremoving an upstream regulator oftranscription. Ineverycase,thesame multiple transcripts were observed, withtheir 5'endslocated downstream ofthenormalHSV-1tktranslation initiation codon. We conclude thatthetranscription ofHSV-1tkinthetestis isdirected bya cryptic TATA box-independent promoter located inthecoding region ofthegene.Thelongest HSV-1thymidine kinase (TK) polypeptides synthesized inthetestis were shorter thanfull-length TK andprobably result fromtranslational initiation atMet46 andMet6o, thesecond andthird ATG codons ofthetkreading frame. Malemiceofmost transgenic lines were sterile, andtheseverity ofthelesion inspermatogenesis was directly related tothelevel ofTK expression. Inthemosthighly expressing lines, sperm counts were lowandmorphologically defective sperm were common. Inother sterile lines, TK was expressed atalowerlevel andsperm counts were normal butspermmotility was greatly reduced. Lineswiththelowest levels ofHSV-1TK expression were fertile. HSV-1TK was expressed ingerm line cells, mainly inthehaploid spermatids. However, low-level HSV-1TK activity was foundinthetestis before thefirst germ cells entered meiosis, showing thatifexpression isconfined tothegerm cells, italso occursinspermatogonia.
Heart disease is a pressing public health problem and the leading cause of death worldwide. The heart is the first organ to gain function during embryogenesis in mammals. Heart development involves cell determination, expansion, migration, and crosstalk, which are orchestrated by numerous signaling pathways, such as the Wnt, TGF-β, IGF, and Retinoic acid signaling pathways. Human-induced pluripotent stem cell-based platforms are emerging as promising approaches for modeling heart disease in vitro. Understanding the signaling pathways that are essential for cardiac development has shed light on the molecular mechanisms of congenital heart defects and postnatal heart diseases, significantly advancing stem cell-based platforms to model heart diseases. This review summarizes signaling pathways that are crucial for heart development and discusses how these findings improve the strategies for modeling human heart disease in vitro.
The distribution of the c-myc protein was studied in the developing embryo from the two-somite stage to embryonic day 17 (E17). A triple labelling method was used, with a polyclonal serum recognizing the human and avian c-myc proteins as the first marker followed by Hoechst 33258 for nuclear staining and the monoclonal antibody 13F4 which reveals the avian myogenic lineage. In situ hybridization was carried out at three selected stages (E3, E6 and E8), in order to compare the distribution of myc mRNA and myc protein. The c-myc protein signal was barely detectable in blastodisc nuclei during the period of somlte formation, after which it became ubiquitous in the embryonic body until E4. Myotomal cell nuclei displayed a strong signal until their organization Into premuscular masses.
On day 4, the level of c-myc protein decreased In all embryonic tissues. By doubling the antibody titre and amplifying the signal by means of the streptavidin-blotin method, c-myc could still be detected in nuclei of defined groups of cells. Such was the case in some mesenchyme-derived tissues at critical periods of or-ganogenesis, for instance in prechondrogenic condensations or hemopoietic cell foci at E6, the latter becoming negative at E9. The heart ventricle displayed a patchwork of positive and negative nuclei from E6 to E10. A myc signal restricted to the quail species was found In the wall of the carotid arteries. Cell nuclei in the nervous system displayed a detectable signal which became restricted to postmitotic neurones. In the ectoderm, the c-myc protein was generally not present after E4, except In presumptive feather buds at the time of epitheliomesenchymal interactions. Endodermal cells (such as hepatocytes, oesophageal and tracheal epithelia) did not express detectable levels of c-myc at any time.
Our results reveal a time- and tissue-specific expression of c-myc during avian development. It is noteworthy that the expression of the c-myc protein often appears dissociated from cell proliferation as shown by the absence of the signal in endodennal cells at E3-E13 as well as its presence in postmitotic neurones.
Finally, although RNA and protein are simultaneously detected in some structures such as presumptive feather buds, their expression is dissociated in endodermal tissues, notably hepatocytes, where in situ hybridization detects a large number of RNA copies with no detectable protein signal.
During brain development, neural precursor cells (NPCs) in different brain regions produce different types of neurons, and each of these regions plays a different role in the adult brain. Therefore, precise regionalization is essential in the early stages of brain development, and irregular regionalization has been proposed as the cause of neurodevelopmental disorders. The mechanisms underlying brain regionalization have been well studied in terms of morphogen‐induced expression of critical transcription factors for regionalization. NPC potential in different brain regions is defined by chromatin structures that regulate the plasticity of gene expression. Herein, we present recent findings on the importance of chromatin structure in brain regionalization, particularly with respect to its regulation by Polycomb‐group proteins and chromatin accessibility. For the precise regionalization during development of the mammalian brain, chromatin‐level regulation in neural precursor cells is essential. In this review, we introduce the recent advances in the regulation of morphogen activity by Polycomb‐group proteins and the role of chromatin accessibility in the human fetal brain.
The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development. We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters. A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins. We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP. Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP. We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells. The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.
HEM13 of Saccharomyces cerevisiae encodes coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway. Expression of HEM13 is repressed by oxygen and heme. This study investigated the regulatory pathway responsible for the regulation of HEM13 expression. The transcriptional activator HAP1 is demonstrated to be required for the full-level expression of HEM13 in the absence of heme. It is also shown that the repression of HEM13 transcription caused by heme involves the HAP1 and ROX1 gene products; a mutation in either gene results in derepression of HEM13 expression. The heme-dependent expression of ROX1 was found to require functional HAP1, leading one to propose that repression of HEM13 results from a pathway involving HAP1-mediated regulation of ROX1 transcription in response to heme levels followed by ROX1-mediated repression of HEM13 transcription. In support of this model, expression of ROX1 under control of the GAL promoter was found to result in repression of HEM13 transcription in a hap1 mutant strain. The ability of ROX1 encoded by the galactose-inducible ROX1 construct to function in the absence of HAP1 indicates that the only role of HAP1 in repression of HEM13 is to activate ROX1 transcription.
The mouse Wnt family comprises at least 10 members sharing substantial amino acid identity with the secreted glycoprotein Wnt-1/int-1. Two of these, Wnt-1 and Wnt-3, are implicated in mouse mammary tumor virus-associated adenocarcinomas, although neither member is normally expressed in the mammary gland. These results suggest the presence of active cellular pathways which mediate the action of Wnt-1 and Wnt-3 signals. An understanding of the normal role of these signalling pathways is clearly necessary to comprehend the involvement of Wnt-1 and Wnt-3 in mammary tumorigenesis. We demonstrate here that five Wnt family members are expressed and differentially regulated in the normal mouse mammary gland. In addition, some of these genes are also expressed in both Wnt-1-responsive and nonresponsive mammary epithelial cell lines. We propose that Wnt-mediated signalling is involved in normal regulation of mammary development and that inappropriate expression of Wnt-1, Wnt-3, and possibly other family members can interfere with these signalling pathways.
The mouse Wnt-1 gene plays an essential role in fetal brain development and can contribute to tumorigenesis when activated aberrantly in the mammary gland. The gene encodes secretory glycoproteins associated with the extracellular or pericellular matrix, and it has been proposed that Wnt-1, as well as its Drosophila homolog wingless, may function in intercellular signalling. We show here that fibroblasts expressing Wnt-1 protein, although not transformed themselves, are able to elicit morphological transformation of neighboring C57MG mammary epithelial cells in coculture experiments. Heparin inhibits this effect, possibly by displacing Wnt-1 protein from its normal site of action. Our results indicate that the Wnt-1 gene can act via a paracrine mechanism in cell culture and strongly support the notion that in vivo the gene may function in cell-to-cell communication.
The proto-oncogene int-1 is activated by adjacent insertions of proviral DNA in mouse mammary tumor virus-induced tumors and has transforming activity in certain mammary epithelial cell lines. The gene is normally expressed in the central nervous system of mid-gestational embryos and in the adult testis. We raised antibodies against synthetic int-1 peptides and used these to identify protein products of the gene in cells transfected or infected with retroviral vectors expressing int-1. Four protein species of 36,000, 38,000, 40,000, and 42,000 Mr were immunoprecipitated by antibodies against two different int-1 peptides and were not present in control cells. Partial degradation with V8 protease showed the four species to be structurally related to each other and to int-1 polypeptide synthesized in vitro. Treatment of the cells with tunicamycin prevented the appearance of all but the 36,000-Mr species, suggesting that the slower-migrating forms are glycosylated derivatives. The unglycosylated 36,000-Mr species migrated faster in polyacrylamide gels than the in vitro translation product of int-1 and has probably undergone cleavage of an amino-terminal signal peptide.
The int-1 proto-oncogene encodes a primary product of 370 amino acids, is normally expressed in mid-gestational embryos and adult testis, and is activated by proviral insertions during mammary carcinogenesis. Polyclonal and monoclonal antibodies directed against int-1-specific synthetic peptides immunoprecipitate up to five forms of int-1 protein, ranging in size from 36,000 to 44,000 Mr, from cell lines that express cloned int-1 DNA introduced by transfection or infection with retroviral vectors. Pulse-chase labeling experiments and glycosidase digestions suggested that the smallest of the int-1 proteins is the primary translation product lacking its signal peptide and that it is modified to produce the larger species of sequential glycosylation. Subcellular fractionations demonstrated that all immunoprecipitable forms of int-1 are mainly associated with membranes. int-1 proteins in crude microsomal preparations are resistant to proteolysis and extractable at elevated pH, suggesting that they are sequestered within cytoplasmic vesicles in a manner consistent with the behavior of secretory products. However, we were unable to identify secreted int-1 products in extracellular fluids.
The Wnt-1 proto-oncogene is essential for proper development of the midbrain and is expressed in a spatially and temporally restricted manner during central nervous system development in mice. In vitro, the gene is specifically transcribed during the retinoic acid (RA)-induced neuroectodermal differentiation of the P19 line of embryonal carcinoma cells. The P19 cells differentiate into neurons, astrocytes, and fibroblast-like cells when treated with RA. Treatment of the cells with dimethyl sulfoxide leads to differentiation along mesodermal lineages, including skeletal and cardiac muscle. We have used the P19 cell line to study the Wnt-1 promoter and identify and characterize the transcription factor(s) that regulates the differentiation-specific transcription of Wnt-1 in RA-treated P19 cultures. Transient-transfection assays have revealed that a 230-bp region comprising positions -278 to -47 of the 5' upstream Wnt-1 sequence was sufficient to direct RA-specific transcription. This promoter fragment was shown to contain a binding site for a nuclear factor that was not detected in undifferentiated P19 stem cells or their dimethyl sulfoxide-treated derivatives but was induced in differentiating RA-treated cells. This factor was termed Wnt-1-inducing factor-1 (WiF-1). DNase I footprinting analysis has identified the G/C-rich WiF-1 binding site, and UV cross-linking studies have shown that WiF-1 is a protein with an M(r) of 65,000. WiF-1 binding activity was also detected in postpubertal mouse testis, the only tissue that expresses Wnt-1 in adults. Site-directed mutations that inhibited WiF-1 binding to the Wnt-1 promoter concomitantly abolished the activity of the promoter in RA-treated P19 cells. The active WiF-1 protein was purified by DNA affinity chromatography. Our data suggest that WiF-1 is a novel G/C box-binding transcription factor and support a physiological role for WiF-1 in the developmentally regulated expression of Wnt-1.
ferT is a testis-specific transcript of FER encoding a truncated version of the potential tyrosine kinase. Using in situ hybridization analysis, we found that ferT was transiently expressed during spermatogenesis and that expression was restricted to spermatocytes at the pachytene stage of meiotic prophase. This pattern of expression is unprecedented by other tyrosine kinases and suggests a role for ferT in a particular stage of spermatogenesis.
A cDNA for a potential tyrosine kinase-encoding mRNA was isolated from a mouse testis cDNA library. In a survey of eight mouse tissues, a transcript of 2.4 kilobases restricted to testis tissue was found. The mRNA encodes a 453-amino-acid protein of 51,383 daltons, the smallest tyrosine kinase protein ever described. RNA synthesized from the cDNA template directs the synthesis of a 51,000-Mr protein in a cell-free translation system. The carboxy-terminal 409 amino acids are 98 and 90% identical to the carboxy halves of the rat and human Fer proteins, respectively. This suggests that the cDNA represents an alternatively spliced testis-specific fer mRNA and is therefore termed by us ferT. On the basis of the appearance time of the fer mRNA in the testis of maturing neonatal mice, we speculate on the role played by this protein in the development of this organ.
Wnt-1 (int-1) is a cellular oncogene often activated by insertion of proviral DNA of the mouse mammary tumor virus. We have mapped the 5' end and the promoter area of the Wnt-1 gene by nuclease protection and primer extension assays. In differentiating P19 embryonal carcinoma cells, in which Wnt-1 is naturally expressed, two start sites of transcription were found, one preceded by two TATA boxes and one preceded by several GC boxes. In P19 cells, a 1-kilobase upstream sequence of Wnt-1 was able to confer differentiation-specific expression on a heterologous gene. We have investigated how Wnt-1 transcription was affected by mouse mammary tumor virus proviral integrations in various configurations near the promoters of the gene. One provirus has been inserted in the 5' nontranslated part of Wnt-1, in the same transcriptional orientation, and has functionally replaced the Wnt-1 promoters. Wnt-1 transcription in this tumor starts in the right long terminal repeat of the provirus, with considerable readthrough transcription from the left long terminal repeat. Another provirus has been inserted in the orientation opposite that of Wnt-1 into a GC box, disrupting the first Wnt-1 transcription start site but not the downstream start site. Most insertions have not structurally altered the Wnt-1 transcripts and have enhanced the activity of the normal two promoters.
The murine int-1 proto-oncogene has been implicated in neural development and, when transcriptionally activated by mouse mammary tumor virus, contributes to the genesis of mammary tumors. To understand the function of the int-1 gene product in these processes, we have characterized the biochemical properties of int-1 protein expressed in a neuroendocrine cell line transfected with int-1 cDNA. Here we provide evidence that int-1 protein is secreted and associates with the cell surface. int-1 protein was very efficiently processed and secreted through the constitutive secretory pathway, although no int-1 protein could be immunoprecipitated from the culture medium. Treatment with suramin effectively released mature int-1 proteins into the culture fluid, which suggests that secreted int-1 protein associates with the cell surface or extracellular matrix. We have also shown directly, by radioiodination of intact cells and by surface antibody adsorption, that secreted int-1 proteins can be detected on the cell surface. These data support a model in which int-1 protein is secreted and functions locally in cell-to-cell signaling.
We reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase reporter gene (tk) was expressed in the testes of transgenic mice when coupled to the promoter of a liver-specific mouse major urinary protein (MUP) gene. Here we show that HSV-1 tk is also expressed in the testis when coupled to a MUP pseudogene promoter, to a truncated MUP promoter that is not active in the liver, and to the promoter of the bovine thyroglobulin gene. Furthermore, HSV-1 tk itself was expressed in the testis, although its normal expression had been disabled by removing an upstream regulator of transcription. In every case, the same multiple transcripts were observed, with their 5' ends located downstream of the normal HSV-1 tk translation initiation codon. We conclude that the transcription of HSV-1 tk in the testis is directed by a cryptic TATA box-independent promoter located in the coding region of the gene. The longest HSV-1 thymidine kinase (TK) polypeptides synthesized in the testis were shorter than full-length TK and probably result from translational initiation at Met46 and Met60, the second and third ATG codons of the tk reading frame. Male mice of most transgenic lines were sterile, and the severity of the lesion in spermatogenesis was directly related to the level of TK expression. In the most highly expressing lines, sperm counts were low and morphologically defective sperm were common. In other sterile lines, TK was expressed at a lower level and sperm counts were normal but sperm motility was greatly reduced. Lines with the lowest levels of HSV-1 TK expression were fertile. HSV-1 TK was expressed in germ line cells, mainly in the haploid spermatids. However, low-level HSV-1 TK activity was found in the testis before the first germ cells entered meiosis, showing that if expression is confined to the germ cells, it also occurs in spermatogonia.
We isolated and sequenced a cDNA clone of the human gene encoded by the 5' half of the ret transforming gene. The nucleotide sequence indicates that it encodes a protein with "finger" structures which represent putative metal- and nucleic acid-binding domains. Transcription of this gene was detected at high levels in a variety of human and rodent tumor cell lines, mouse testis, and embryos. In addition, a unique transcript was observed in testis RNA. When the expression of the unique transcript was examined at different stages of spermatogenesis, a striking increase in mRNA levels accompanied progression from meiotic prophase pachytene spermatocytes to postmeiotic round spermatids. This finger-containing gene may thus function in male germ cell development.
The int-1 proto-oncogene is a target for insertional activation of transcription by mouse mammary tumor virus in many murine mammary tumors. Whereas no expression of int-1 is seen in normal mammary tissue, int-1 RNA can be detected in normal mice in the neural tubes of midgestation embryos and in postmeiotic spermatocytes from adult testes. I report here the results of a study in which several different antibodies against synthetic peptides were produced and used to characterize the processing and secretion of int-1 protein. CHO cells were transfected with an inducible int-1 expression vector that was subsequently amplified to generate cell lines expressing very high levels of int-1 protein. Immunoprecipitation of [35S]cysteine-labeled cell lysates from these CHO cells yielded large amounts of four immature forms of int-1 glycoprotein (molecular weights of 36,000, 38,000, 40,000, and 42,000). A significant fraction of these int-1 species formed disulfide-linked multimers. Pulse-chase and glycosidase digestion studies demonstrated that some of the immature species of int-1 protein move through the secretory pathway and are processed to a mature heterogeneous glycoprotein with a molecular weight of about 44,000. Suramin treatment of the CHO cells during pulse-chase experiments increased the amount of 44,000-molecular-weight int-1 protein in the culture medium.
Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.
In mouse embryos, the int-1 proto-oncogene is transiently expressed in areas of the developing neural system. Retinoic acid-treated P19 embryonal carcinoma cells have often been used as an in vitro model for the molecular basis of neural development. We shown here that int-1 is transiently expressed in differentiated P19 cells. The time course and retinoic acid dose dependence of int-1 expression suggest that the gene is specifically expressed during early neural differentiation. P19 cells may be a useful model to assist in the study, at the cellular level, of the role of int-1 in neural development.
Agonist-promoted down-regulation of β-adrenergic receptor mRNA was investigated in S49 mouse lymphoma variants with mutations in elements of hormone-sensitive adenylate cyclase. In wild-type cells steady-state levels of β-adrenergic receptor mRNA were established by DNA-excess solution hybridization to be 1.72 ± 0.08 (n = 8) amol/microgram total cellular RNA. Receptor mRNA levels declined 35–45% in response to stimulation by the β-adrenergic agonist (-)isoproterenol or forskolin as described previously in DDT1 MF-2 cells (Hadcock, J. R., and Malbon, C. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5021–5025). Agonist-promoted cAMP accumulation and down-regulation of receptor mRNA were analyzed in three variants with mutations in Gsα (H21a, unc, cyc⁻) and a single variant lacking cAMP-dependent protein kinase activity (kin⁻). H21a (Gsα coupled to receptor, but not to adenylate cyclase), unc (Gsα uncoupled from receptor), and cyc⁻ (lacking Gsα) variants accumulated cAMP and down-regulated β AR mRNA in response to forskolin. In unc and cyc⁻ cells isoproterenol failed to stimulate cAMP; accumulation and down-regulation of receptor mRNA was not observed. H21a cells, in contrast, displayed agonist-promoted regulation of β-adrenergic receptor mRNA but only basal levels of cAMP accumulation in response to isoproterenol. The kin- cells displayed cAMP accumulation in response to forskolin as well as to isoproterenol but no down-regulation of receptor mRNA or receptor expression. Taken together these data demonstrate several features of agonist-promoted down-regulation of mRNA: (i) cAMP-dependent protein kinase activity is required for down-regulation of mRNA (kin⁻), although elevated cAMP accumulation is not (H21a); (ii) functional receptor-Gs coupling is required (H21a), and clones lacking Gsα (cyc⁻) or receptor Gs coupling (unc) lack the capacity to down-regulate mRNA in response to agonist; and (iii) in the presence of basal levels of cAMP and cAMP-dependent protein kinase activity, functional receptor-Gs coupling (H21a) to some other effector other than adenylate cyclase may be propagating the signal.
Gastrulation occurs between 6.5 and 8.0 days post coitum in the mouse and is associated with a dramatic increase in cell proliferation, being initiated when the embryo consists of some 500-600 cells and resulting in a neural plate stage embryo containing 50,000 cells or more. There is evidence to suggest that some of these areas may be determined with respect to their future development quite early in the process of gastrulation. During this period of development it is clear that the growth rate of the whole embryo, and of its constituent parts, is under intrinsic control and capable of controlled variation aimed presumably at generating an organogenesis stage embryo of a notional target size in which the parts are of appropriate relative size. Investigations in the mouse have shown that growth of the embryo parts and their co-ordination can be profoundly disturbed in early embryonic stages by insult with the cytotoxic agent, Mitomycin C. MMC is an alkylating agent that inhibits DNA synthesis. It causes cell death and reduces pre-organogenesis stage embryos to 10% of normal size as measured by cell number. During the progression of embryogenesis the normal co-ordination of development of different parts of the embryo is abnormal. Considerable and localized growth control is obviously operating in these stages, thus the proto-oncogenes with known growth regulatory functions were looked at as likely candidates controlling the dramatic growth patterns observed. During the gastrulation movements the primordial germ cells are located at the base of the allantois and appear to 'escape' the movements and rearrangements of gastrulation by migrating into the extraembryonic membranes. The primordial germ cells then appear to undergo their own differentiation, proliferate and migrate into the genital ridges. The germ cells multiply as they migrate until some 5000 cells ultimately invade the gonads. The proto-oncogene c-kit expression was analysed, as it has been recently identified with the dominant white spotting (W) locus of the mouse. The W mutations have pleiotropic effects on mouse development, one of which is to cause an impairment of proliferation and/or migration of primordial germ cells. It appears that the c-kit product is necessary for the maintenance of the primordial germ cells during their migratory/mitotically active phase. This finding holds true for overall expression of c-kit in structures of the developing embryo. As the testes develop and become functional, the germ cells undergo further differentiation. There is evidence from Y chromosome deletion mapping that there is a multiple copy gene on the long arm of the mouse Y which is needed for the normal development of the sperm head. A Y-specific genomic DNA sequence (Y353/B) has previously been described which is present in multiple copies on the long arm of the mouse Y and is specifically transcribed in the testis. Using quantitative Northern analysis it was shown that the RNA transcripts are from the multiple copies. In situ hybridization was used to demonstrate that the transcripts are In the germ line and not In the soma, and that the expression is predominantly in round spermatids. This was the first evidence for a Y chromosome gene being transcribed in the testis (and probably anywhere). Y353/B is thus a candidate for being the multiple copy 'sperm morphology gene' on the mouse Y chromosome.
This FlyBook chapter summarizes the history and the current state of our understanding of the Wingless signaling pathway. Wingless, the fly homolog of the mammalian Wnt oncoproteins, plays a central role in pattern generation during development. Much of what we know about the pathway was learned from genetic and molecular experiments inDrosophila melanogaster, and the core pathway works the same way in vertebrates. Like most growth factor pathways, extracellular Wingless/Wnt binds to a cell surface complex to transduce signal across the plasma membrane, triggering a series of intracellular events that lead to transcriptional changes in the nucleus. Unlike most growth factor pathways, the intracellular events regulate the protein stability of a key effector molecule, in this case Armadillo/β-catenin. A number of mysteries remain about how the "destruction complex" destabilizes β-catenin and how this process is inactivated by the ligand-bound receptor complex, so this review of the field can only serve as a snapshot of the work in progress.
The processes of cell transformation in tissue culture and neoplastic transformation in vivo produce cells which are recognizable by their distinctive morphology and by their ability to proliferate under conditions which do not support, or severely limit, the proliferation of their normal counterparts. The altered morphology is recognized by the pathologist in tumor biopsies and is used not only for diagnosis of malignancy but also for tumor staging and prognostic prediction. In tissue culture the altered cell morphology is recognized as foci of cells which are morphologically distinct from the parental-type cells which surround the focus. This has been used for the development of assays for oncogenic viruses (Temin and Rubin 1958) and for the identification of oncogenes derived from tumors and introduced into the indicator cells by DNA transfection (Shih et al. 1979; Cooper 1982). In both cases the diagnosis or identification of the altered cells may be aided by the excess proliferation of these cells in relation to their normal counterparts. The altered morphology is an indication of changes in the synthesis of products which allow us to distinguish cell types. Typically, these are not products required for cell survival, although the synthesis of “house-keeping” products may be quantitatively altered. They reveal alterations in cell-type specific products or developmentally regulated products. Thus, the morphological changes are indicators of changes affecting the differentiation of the cell. This idea is strengthened by analysis of the synthesis of these differentiated cell products using biochemical and molecular biological techniques.
Infertility is generally defined as a failure to conceive following one year of unprotected intercourse; it affects about 10% to 15% of couples attempting a pregnancy for the first time (Romeny et al., 1975). Abnormalities in the male partner contribute to infertility in about 40% of these couples (Speroff et al., 1983).
The seminal discovery that the ability of retro-viruses to transform normal cells into malignant cells is due to the incorporation of cellular genes into the viral genome1 led to a new era of thinking about cancer. It became clear that malignant transformation was not the consequences of acquiring new genes, but the result of alterations in existing cellular gens. These findings heralded new ways of investigating malignant cells and, perhaps even more important, provided support for the concept that the similarities between malignant cells and embryonic cells are due to expressions of the same gene programs.
Current molecular biology technology provides an increased level of sensitivity for the analysis of sperm structure and function. Excellent laboratory manuals are available that provide familiarity with the few basic concepts required for mastery of these techniques (Maniatis et al.,1982; Ausubel et al.,1987; Berger and Kimmel, 1987). Therefore, this chapter does not detail such laboratory procedures but rather attempts to review the recent literature in which sperm function has been studied by using the tools of molecular biology. Basically, I focus on the investigations that involve DNA and RNA isolation, cloning, and analysis to shed light on the mechanisms whereby the spermatozoan acquires the unique properties necessary to achieve success in accomplishing its primary function, fertilization of the egg. This overview is intended to be instructive rather than exhaustive, and I hope it will provide a resource with which to initiate further study of fertilization.
Recent developments of experimental techniques in cellular and molecular biology have made it possible to understand the molecular biology of male gametogenesis in greater detail. This book focuses on the description of specialized proteins, which are dominantly and/or specifically expressed in germ cells and localized in spermatozoa. There is an urgent need to classify proteins of spermatogenic cells with a view of their functions, and their applications in the regulation of fertility and in understanding infertility. The understanding of structural properties of male germ cell specific proteins can offer vulnerable points for targeted intervention in testis without generalized effects on stages of spermatogenesis. Besides targeted action in male germ cells, sperm specific proteins and polypeptides may also offer potential application in the development of a contraceptive vaccine. Researchers in the fields of biochemistry, cell biology, molecular biology, reproductive biology and proteomics will find this book of interest. "This book is far superior to any previous book on the subject. I recommend this book with great enthusiasm." © 2005 Springer Science+Business Media, Inc. All rights reserved.
In recent years we have witnessed a revolution in the understanding of the mechanisms which regulate mammalian cell growth. This applies both to normal cells, in which growth is tightly controlled, as well as to cancer cells, which divide in an uncontrolled fashion. With the advent of molecular biology a number of genes have been identified whose products are involved in regulating normal cell growth. In parallel research 40–60 genes that are capable of inducing a transformed phenotype and that have been termed “on-cogenes” have been identified. It is now clear that growth control genes on the one hand and oncogenes on the other are largely one and the same. Although unequivocal evidence for this notion was obtained only recently, the concept itself emerged gradually beginning with the discovery of viral oncogenes (v-onc genes) and the fact that these genes represent cell-derived sequences (termed protooncogenes, cellular oncogenes, or c-onc genes).
The proto-oncogene int-1 is implicated in mouse mammary tumour formation and encodes a protein of unknown function that is probably secreted. int-1 has a highly restricted pattern of normal expression, limited to the mature testis and a transient expression during mouse development. To gain clues as to the normal function of int-1 we have examined the embryonic sites of int-1 RNA accumulation by in situ hybridization. int-1 expression is restricted to a subset of cells in the developing central nervous system. In the spinal cord, these cells comprise the non-neuronal roof plate. Possible functions of int-1 in the organization of the mouse neural tube are presented in light of the recent finding that the Drosophila homologue of int-1 is wingless, a segment polarity gene.
The Wnt-1 gene plays an essential role in fetal brain development and encodes a secreted protein whose signaling mechanism is presently unknown. In this report we have investigated intracellular mechanisms by which the Wnt-1 gene induces morphological changes in PC12 pheochromocytoma cells. PC12 cells expressing Wnt-1 show increased steady-state levels of the adhesive junction protein plakoglobin, and an altered distribution of this protein within the cell. This effect appears similar to a modulation of the plakoglobin homolog, Armadillo, that occurs in Drosophila embryos in response to the Wnt-1 homolog, wingless (Riggleman, B., P. Schedl, and E. Wieschaus. 1990. Cell. 63:549-560). In addition, PC12/Wnt-1 cells show elevated expression of E-cadherin and increased calcium-dependent cell-cell adhesion. These results imply evolutionary conservation of cellular responses to Wnt-1/wingless and indicate that in certain cell types Wnt-1 may act to modulate cell adhesion mechanisms.
Proteins encoded by the Wnt family of genes act as signals and have been shown to play important roles in a wide variety of developmental processes. Here we describe the cloning of three Wnt family members from the zebrafish, Danio rerio, which encode proteins with homology to murine Wnt-2, -4 and -5A/B. The expression patterns of the latter two zebrafish genes, designated ZfWnt4 and ZfWnt5 show considerable similarity with their homologues in other vertebrates; ZfWnt2, however, is expressed in the developing viscera in a pattern distinct from its closest murine homologue. In the light of the similarities and differences in the patterns of expression of these genes relative to their homologues in other vertebrates, we speculate on their possible functions.
Spermatogenic genes are those genes that are newly expressed or whose expression is augmented in spermatogonia, spermatocytes or spermatids. They include: (1) genes expressed exclusively during spermatogenesis, (2) testis-specific isozymes, isotypes and variants and (3) somatic genes showing increased levels of expression in testis. Molecular-genetic analysis is beginning to provide information about the control and expression of spermatogenic genes.
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Weisolated andsequenced acDNAclone ofthehumangeneencoded bythe5'half oftherettransforming gene. Thenucleotide sequence indicates thatitencodes aprotein with"finger" structures whichrepresent putative metal- andnucleic acid-binding domains. Transcription ofthis genewasdetected athighlevels ina variety ofhumanandrodent tumorcell lines, mousetestis, andembryos. Inaddition, aunique transcript was observed intestis RNA.Whentheexpression oftheunique transcript wasexamined atdifferent stages of spermatogenesis, a striking increase inmRNA levels accompanied progression frommeiotic prophase pachytene spermatocytes topostmeiotic roundspermatids. Thisfinger-containing genemaythusfunction in malegermcell development.
A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).
A clone, pHBT-1, containing sequences homologous to Drosophila homeo boxes has been isolated from a mouse testis cDNA library. The sequence is 80% homologous at the DNA level and 88% homologous at the amino acid level to the homeo box sequence of the Antennapedia gene of Drosophila. Sequences flanking the 3' end of the homeo box are highly diverged from other murine homeo box-containing genes characterized to date. RNA blot hybridization analysis of mouse testis poly(A)+ RNA revealed transcripts of approximately 1.4 kb in length. Within the limits of sensitivity of detection of Northern blot analysis, no transcripts were seen in any of the adult somatic tissues examined. Other tissues that contain stem cells, namely those of the hemopoietic system, also lacked detectable amounts of HBT-1 transcripts. HBT-1 transcripts were limited to male germ cell-containing tissues, since RNAs from juvenile and adult ovaries did not contain detectable amounts of the 1.4-kb transcripts. Expression of the HBT-1 gene was not detected in embryonic testes, nor in tests of neonatal animals which contain germ cells up to the Type B stage of spermatogonial development. A role for the expression of the HBT-1 gene in the meiotic stages of male germ cell differentiation is postulated.
Hox-1.4 is a mouse homeobox-containing gene (initially identified as HBT-1), whose expression appears to be testis-specific in the adult animal. Examination of Hox-1.4 transcripts in RNA from testes of mutant mice deficient in germ cells confirms that Hox-1.4 expression within the testis is germ cell-specific. Enriched populations of spermatogenic cells were used to localize the expression of Hox-1.4 specifically to germ cells that have entered into and progressed beyond the meiotic prophase stage of differentiation and to demonstrate the presence of two different size Hox-1.4 transcripts. Examination of RNA from teratocarcinoma cell cultures and mouse embryos at 10.5-16.5 days of gestation demonstrated the presence of several Hox-1.4 transcripts, which are larger than those present in germ cells. In the midgestation fetus, Hox-1.4 expression is most abundant in the spinal cord.
The mouse int-1 gene is a putative mammary oncogene discovered as a target for transcriptionally activating proviral insertion
mutations in mammary carcinomas induced by the mouse mammary tumor virus in C3H mice. We have isolated molecular clones of
full- or nearly full-length cDNA transcribed from int-1 RNA (2.6 kilobases) in a virus-induced mammary tumor. Comparison of
the nucleotide sequence of the cDNA clones with that of the int-1 gene (A. van Ooyen and R. Nusse, Cell 39:233-240, 1984)
shows the following. The coding region of the int-1 gene is composed of four exons. The splice donor and acceptor sites conform
to consensus; however, at least two closely spaced polyadenylation sites are used, and the transcriptional initiation site
remains ambiguous. The major open reading frame is preceded by an open frame 10 codons in length. The mRNA encodes a 41-kilodalton
protein with several striking features--a strongly hydrophobic amino terminus, a cysteine-rich carboxy terminus, and four
potential glycosylation sites. There are no differences in nucleotide sequence between the known exons of the normal and a
provirally activated allele. The length of the deduced open reading frame was further confirmed by in vitro translation of
RNA transcribed from the cDNA clones with SP6 RNA polymerase.
A new murine homeo-box (Hox1-3) has been isolated and studied with respect to its structure and transcriptional pattern. This homeo-box is part of a gene which is specifically regulated during mouse prenatal development and expressed in a restricted number of teratocarcinoma tumours as well as in adult testis. Hox1-3 is shown to be a member of a cluster of at least four homeo-boxes lying within a 75-kb segment of DNA on mouse chromosome 6. The structure of the whole cluster, the Hox-1 locus is presented.
The int-1 gene is often activated by proviral insertion in mouse mammary tumors. Direct evidence for the normal function of this gene and its role in tumorigenesis has therefore been lacking. To examine possible biological effects of int-1 activation in in vitro cell systems, we have constructed recombinant molecules of genomic int-1 DNA, transcriptionally activated by retroviral promoters. Transfection of these constructs into cuboidal RAC311C mammary cells leads to morphological transformation of the cells and rapid tumorigenicity.
c-mos RNA transcripts have been previously detected in mouse gonadal tissue and in late-term embryos. Here, we show that they are also present at low levels in placenta and in adult mouse brain, kidney, mammary gland, and epididymis. Marked differences are observed in the size of the mos RNA transcripts detected in different tissues. All transcripts appear to end at the same 3' position, and the tissue-specific size variations appear to be due to the use of different promoters. For example, the testicular and ovarian RNA transcripts initiate approximately 280 and approximately 70 base pairs, respectively, upstream from the first initiation codon, but both end at a common site downstream from the mos open reading frame. The expression of mos is developmentally regulated in gonadal tissue. Thus, the level of mos transcripts in testes is low for the first 3 weeks after birth, increases at least 10-fold around day 25, and reaches adult levels by day 30. In contrast, ovaries from preweaning mice contain a higher level of mos mRNA compared to ovaries from adult mice. In cell fractionation experiments we show that mos transcripts are present in haploid germ cells. We find that these transcripts are associated with monosomes and polysomes. The peculiar pattern of mos expression in mouse gonadal tissue suggests a role for the c-mos proto-oncogene in germ cell differentiation.
The protooncogene c-mos is expressed in murine reproductive tissues, producing transcripts of 1.7 and 1.4 kilobases in testis and ovary, respectively. In situ hybridization analysis of c-mos expression in histological sections of mouse ovaries revealed that oocytes are the predominant if not exclusive source of c-mos transcripts. c-mos transcripts accumulate in growing oocytes, increasing 40- to 90-fold during oocyte and follicular development. c-mos transcripts were also detected in male germ cells and are most abundant after the cells have entered the haploid stage of spermatogenesis. This developmentally regulated pattern of c-mos expression in oocytes and spermatogenic cells suggests that the c-mos gene product may have a function in normal germ-cell differentiation or early embryogenesis.
Five mouse alpha-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within the coding region. Most, though not all of these isotype-specific amino acids, are clustered at the carboxy terminus. One of the alpha-tubulin isotypes described is expressed exclusively in testis and is encoded by two closely related genes (M alpha 3 and M alpha 7) which have homologous 3' untranslated regions but which differ at multiple third codon positions and in their 5' untranslated regions. We show that a subfamily of alpha-tubulin genes encoding the same testis-specific isotype also exists in humans. Thus, we conclude that the duplication event leading to a pair of genes encoding a testis-specific alpha-tubulin isotype predated the mammalian radiation, and both members of the duplicated sequence have been maintained since species divergence. A second alpha-tubulin gene, M alpha 6, is expressed ubiquitously at a low level, whereas a third gene, M alpha 4, is unique in that it does not encode a carboxy-terminal tyrosine residue. This gene yields two transcripts: a 1.8-kilobase (kb) mRNA that is abundant in muscle and a 2.4-kb mRNA that is abundant in testis. Whereas the 1.8-kb mRNA encodes a distinct alpha-tubulin isotype, the 2.4-kb mRNA is defective in that the methionine residue required for translational initiation is missing. Patterns of developmental expression of the various alpha-tubulin isotypes are presented. Our data support the view that individual tubulin isotypes are capable of conferring functional specificity on different kinds of microtubules.
Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in
extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence
was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb
actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round
and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA,
pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of
a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations
with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower
amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that
round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes
hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.
Quaking (qk), an autosomal recessive mutation previously reported to cause deficient myelination in the central nervous system, is now shown to cause virtual azoöspermia in homozygous male mice. Kinetic analysis of spermatogenesis indicates that the defect is one of spermatid differentiation, not of production. Electron microscopic examination of spermiogenesis in the mutant demonstrates two principal morphogenetic defects. (1) As chromatin condenses the nucleus and structures surrounding it assume abnormal shapes. Extensions of the adjacent Sertoli cell indent the nuclear space of the spermatid. (2) The axoneme of the flagellum becomes disorganized. Many normal features of spermatid ultrastructure, including some that have not previously been described in the mouse, e.g., the mortise and a substructure of the acrosome’s equatorial segment, differentiate normally in affected spermatids. The multiple effects of the qk mutation may be mediated by a single biochemical abnormality, perhaps of lipid metabolism, but the qk locus is near a complex region, T, known to carry other genes influencing sperm morphogenesis and motility; the possibility is raised that qk is interacting with neighboring loci and may exert its pleiotropic effects by more than one mechanism.
Unique DNA sequences can be determined directly from mouse genomic DNA. A denaturing gel separates by size mixtures of unlabeled DNA fragments from complete restriction and partial chemical cleavages of the entire genome. These lanes of DNA are transferred and UV-crosslinked to nylon membranes. Hybridization with a short 32P-labeled single-stranded probe produces the image of a DNA sequence "ladder" extending from the 3' or 5' end of one restriction site in the genome. Numerous different sequences can be obtained from a single membrane by reprobing. Each band in these sequences represents 3 fg of DNA complementary to the probe. Sequence data from mouse immunoglobulin heavy chain genes from several cell types are presented. The genomic sequencing procedures are applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution.
A method for obtaining highly purified fractions of rat testicular cells is described. Single cell suspensions from adult rat testes were separated by centrifugal elutriation. Fractions enriched in pachytene primary spermatocytes, early spermatids, and cytoplasts detached from late spermatids were obtained. These fractions were further separated by equilibrium density centrifugation on gradients of Percoll. In this manner fractions of 3 x 10⁷ pachytene spermatocytes (98% purity), 1.1 x 10⁸ early spermatids (93% purity), and 1.1 x 10⁸ cytoplasts (98% purity) were obtained within 6 h after sacrificing the rats. The cells appeared to be morphologically intact and to have retained their biochemical integrity.
Analysis of acid-soluble nuclear proteins by polyacrylamide gel electrophoresis showed that histone 4 is synthesized during the pachytene stage, and confirmed that testis-specific histones are also synthesized during this stage. Analysis of a microsomal RNA preparation from purified pachytene spermatocytes and purified early spermatids by sucrose gradients indicated that intact ribosomal RNA (rRNA) can be obtained from purified cells. Both cell types are active in synthesizing presumptive messenger RNA (mRNA) with a wide range of sedimentation values, but no appreciable rRNA synthesis was detected.
The acutely oncogenic retroviruses induce a wide spectrum of malignancies including sarcomas, carcinomas, lymphomas and leukaemias1. They all contain sequences which are required for neoplastic transformation as an integral part of their genomes1–3. These sequences, termed viral oncogenes (v-onc), apparently originated from the normal vertebrate genome.To date, cellular homologues of over a dozen different oncogenes of acutely oncogenic viruses have been identified1–4. Although transcription of some c-onc genes has been detected in normal vertebrate cells1,5–7, relatively little is known about their role in normal cell metabolism. We describe here stage- and tissue-specific expression of cellular genes homologous to the oncogenes of FBJ murine osteosarcoma virus, Abelson imirine leukaemia virus and Harvey sarcoma virus during mouse prenatal and early postnatal development. Our results suggest participation of cellular oncogenes in normal developmental processes.
A method utilizing sequential enzymatic incubation in collagenase (1 mg/ml) and trypsin (2.5 mg/ml) has been developed for the dissociation of the seminiferous epithelium. A significant advantage of this method is that, following collagenase incubation and washings in an enriched Krebs-Ringer bicarbonate buffer solution, isolated seminiferous tubules are obtained which are free of interstitial cells. The “purified” seminiferous epithelium is then dissociated with trypsin. A further advantage of this dissociation technique has been a reduction in the number of symplasts (multinucleate cells) which form by the opening up of the intercellular bridges that occur between synchronously differentiating clusters of germ cells. Both the elimination of the interstitial cells and the reduction in the number of symplasts have made possible the recovery of more highly enriched germ cell fractions. The homogeneity of the cell fractions was determined by light and electron microscopy. Integrity of the isolated cells was verified by Trypan blue exclusion and measurement of oxygen consumption.
FAMILIAL hypogonadism in man, due to an isolated deficiency of gonadotrophin secretion, has been well documented1-6, but difficult to investigate because of the lack of a suitable animal model4. We report here the genetic and endocrinological background of a mutant strain of mouse in which the testes and ovaries fail to develop postnatally. The primary cause of this seems to be a deficiency in hypothalamic gonadotrophin-releasing hormone (GnRH) with a consequent reduction in pituitary content and circulating levels of luteinising hormone (LH) and follicle-stimulating hormone (FSH). By analogy with the Brattleboro rat (genetic defect in vasopressin synthesis) this mutant should prove useful for studying the synthesis of hypothalamic releasing hormones as well as the role of the hypothalamic-gonadotrophin system in sexual differentiation, puberty, folliculogenesis and spermatogenesis. The mutant has been named hypogonadal, symbol hpg.
Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 times 10(8) cells, prepared from six mouse testes or from one hanster testis, were separated into 11 fractions in less than two hours as compared to the 4--5 hours required for sedimentation at unit gravity ("Staput"). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1--8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1--8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 times 10(9) cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blud and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.
Neural tissues contain high levels of the cellular homologue of the transforming protein of Rous sarcoma virus (RSV), but neither the specific cell types expressing high levels of c-src, nor the function of the cellular src (c-src) protein has been determined. Using primary culture methods, we have found that pure neurones and astrocytes derived from the rat central nervous system (CNS) contain 15- to 20-times higher levels of the c-src protein than fibroblasts. However, the specific activity of the c-src protein from the neuronal cultures is 6- to 12-times higher than that from the astrocyte cultures. In addition, the c-src protein expressed in neuronal cultures contains a structural alteration within the amino-terminal region of the molecule that causes a shift in the mobility of the c-src protein on the SDS-polyacrylamide gels. These results indicate that a structurally distinct form of the cellular src protein that possesses an activated tyrosylkinase activity is expressed at very high levels in post-mitotic CNS neurones.
A murine homeo box (m6-12) known to be expressed during differentiation of embryonal carcinoma cells lies within a cluster of homeo boxes located in 30 kilobase pairs of genomic DNA. We have established the organization of the boxes within this complex, as well as the nucleotide sequence of a second box, m5-4. Similar to the m6-12 box, expression of novel m5-4 transcripts is induced upon differentiation of embryonal carcinoma cells. Transcripts of genes containing m6-12 were found in embryonic tissue during almost all stages of prenatal development studied, whereas expression of m5-4 was detected only in 12 day embryonic tissue. Finally, we have described the differential expression of these homeo-box-containing regions in various adult tissues.
The int-1 and int-2 genes were first isolated as targets for transcriptional activation by proviral insertion mutations in mammary carcinomas induced by the mouse mammary tumor virus (MMTV). Since these proto-oncogenes are not expressed at detectable levels in previously tested normal tissues from adult mice, we sought to determine whether these genes might be active during embryogenesis by examining mouse embryos and cultured teratocarcinoma cells for RNA encoded by int-1 and int-2. A single size class of int-1 RNA is present only at mid-gestation stages of development (days 8 through 13) and is also detected in testes from postpuberal mice. Four species of int-2 RNA are found in peri-implantation embryos and teratocarcinoma cells and are particularly abundant in derivatives of the primitive endodermal lineage, but int-2 RNA is not detected during mid- or late gestation or in any normal adult tissues tested. Thus, these two proto-oncogenes, activated during mammary carcinogenesis by the same mechanisms, are normally expressed at different times and places in embryonic and adult mice.
Mammalian homeo box genes have been identified on the basis of sequence homology to Drosophila homeotic and segmentation genes. These studies examine the distribution of transcripts from two mouse homeo box genes, Hox-2.1 and Hox-3.1, throughout the latter third of prenatal development. Transcripts from these genes are regionally localized along the rostro-caudal axis of the developing central nervous system, yielding expression patterns very similar to patterns of Drosophila homeotic gene expression.
The homeo box is a 180 bp protein-coding domain found within homeotic genes of Drosophila and conserved in a variety of invertebrate and vertebrate species. It has been suggested that the mammalian homeo box sequences may play a role in controlling pattern formation during embryogenesis. We report findings that support this hypothesis. We have cloned three overlapping recombinant phage clones that cover a region of mouse chromosome 11 that contains a cluster of four homeo boxes (the Hox-2 locus). This locus encodes multiple transcripts that are expressed during embryogenesis. Forty kilobases of the Hox-2 region is devoid of repetitive elements and shows extensive homology with the human Hox-2 locus. These results provide direct evidence for genetic expression during embryonic development, a conserved organization in comparison to the cognate human locus, and a complexity of organization and transcript expression similar to that found in Drosophila.
A common feature of Drosophila homoeo box genes appears to be their spatially restricted expression patterns during morphogenesis. Using Northern blot analysis and in situ hybridization to mouse tissue sections, the spatially restricted expression of a newly identified mouse homoeo box locus, Hox-3, within the central nervous system of newborn and adult mice has been demonstrated.
In mammary tumors induced by the mouse mammary tumor virus (MMTV), the int-1 gene is frequently activated by adjacent proviral insertions and is thereby strongly implicated in tumorigenesis. To seek a direct biological effect of int-1 that would validate its proposed role as an oncogene, we constructed a retrovirus vector containing the gene and examined its effects on tissue culture cells. Expression of int-1 in a mammary epithelial cell line caused striking morphological changes, unrestricted growth at high cell density, and focus formation on a monolayer, although the cells were not tumorigenic in vivo. This partial transformation induced by int-1 was not observed in cells infected by an otherwise identical virus bearing a frameshift mutation in the gene. These findings strongly support the hypothesis that int-1 plays a functional role in MMTV-induced mammary tumorigenesis.
Several genes involved in the determination of Drosophila body segments share a conserved DNA sequence of 183 nucleotides termed the homoeo box. Homologous homoeo box sequences have been detected in the genome of species ranging from insects and anellids to vertebrates, and a number of homoeo box-containing genomic DNA clones have been isolated from Xenopus, mouse and human. We have recently isolated human complementary DNA clones containing homoeo box sequences, representing transcripts from four different genes. We report here the nucleotide sequence of one of these clones (HHO.c10) and show that the corresponding gene is transcribed in human embryos and fetuses at 5-10 weeks post-conception. A major polyadenylated transcript of approximately 2.1 kilobases (kb), as well as RNA species of higher relative molecular mass (Mr), are specifically expressed at a constant level in spinal cord throughout this developmental period.
On the basis of analysis of cDNA clones of alpha-tubulin RNAs expressed during spermiogenesis in chickens, we report the identification
of a novel alpha-tubulin which is expressed exclusively in chicken testes. Comparison of its sequence with those previously
determined not only demonstrates that the encoded polypeptide is significantly divergent from other alpha-tubulins but also
supports the hypothesis that alpha-tubulin isotypes are distinguished by a carboxy-terminal variable region sequence and,
to a lesser extent, by a domain near the amino terminus. Since essentially all previously known alpha-tubulins undergo a unique
cycle of removal and posttranslational readdition of a tyrosine residue at the extreme carboxy terminus, the presence in this
testes alpha-tubulin of a very divergent carboxy terminus that does not contain an encoded tyrosine raises the possibility
that this polypeptide does not participate in the usual cycle of tyrosination/detyrosination.
We have used in situ hybridization and computer-aided reconstruction to study the spatial distribution of expression of the mammary tumor proto-oncogene int-1 during mouse embryogenesis. int-1 RNA accumulation is restricted to specific regions of the neural plate and its derivatives between 9 and 14.5 days of development. int-1 RNA accumulates throughout the neural plate at the anterior head folds of the 9 day embryo but only at its lateral tips in more posterior regions. Following neural tube closure, int-1 expression is restricted to specific regions of the dorsal wall of the brain ventricles and spinal cord, the ventral wall of the midbrain and the diencephalon, and the lateral walls of the neuroepithelium at the midbrain-hindbrain junction. These data suggest that int-1 has a role in the early stages of central nervous system development in the mouse embryo.
The MH-3 gene, which contains a homeo box that is expressed specifically in the adult testis, was identified and mapped to mouse chromosome 6. By means of in situ hybridization with adult testis sections and Northern blot hybridization with testis RNA from prepuberal mice and from Sl/Sld mutant mice, it was demonstrated that this gene is expressed in male germ cells during late meiosis. In the embryo, MH-3 transcripts were present at day 11.5 post coitum, a stage in mouse development when gonadal differentiation has not yet occurred. The MH-3 gene may have functions in spermatogenesis and embryogenesis.
Hybridization of RNA blots of total testicular RNA from prepuberal and sexually mature CD1 mice with several mouse testicular cDNA probes reveals that the mRNA encoding the two mouse protamines, an actin sequence of 1.5 kb, and a post-meiotically expressed 620 nucleotide mRNA are first detected in the testes of mice 22 days of age. These experiments and other studies analysing RNA preparations from isolated populations of testicular cell types with cDNA probes [1, 2] demonstrate that haploid gene expression occurs in the mammalian testis.
Valuable information about proto-oncogenes and their physiological function has been obtained by studying their expression in normal cells. However, expression of the c-mos gene, the cellular homologue of the transforming gene of Moloney murine sarcoma virus, has not been detected in normal mouse cells or tissues. The conservation of the c-mos open reading frame strongly indicates that the gene must function during some portion of the animal life cycle, and other lines of evidence suggested to us that the c-mos proto-oncogene may be expressed at very low levels in normal tissues. We have used a sensitive S1 nuclease assay to screen RNA preparations from mouse tissues and describe here the detection of c-mos-related transcripts especially in mouse embryos, testes and ovaries. The transcripts found in testis RNA are estimated to be approximately 1.7 kilobases (kb) long by Northern analysis. S1 analysis demonstrated that the entire mos open reading frame is present. In contrast, we detect approximately 1.4-kb transcripts in ovary RNA and at least two major transcripts, approximately 2.3 and approximately 1.3 kb, in embryo RNA. The latter transcripts have in common sequences of at least 1 kb, representing most of the c-mos open reading frame. The variation in size of the mos transcript in different tissues suggests a novel regulatory mechanism for the expression of this proto-oncogene.
RNA from immature mouse testes was shown to lack a low-molecular-weight c-abl transcript previously noted to be the predominant
species in adult testes. The developmental pattern of appearance of this c-abl variant was determined by analyzing RNA obtained
from purified populations of testicular cells in different stages of spermatogenesis. The appearance of the c-abl testicular
variant was coincident with the entry of the germ cells into their haploid state and suggested that the regulated expression
of this proto-oncogene may be important in the normal differentiation of the male germ line.
The homoeo box sequence of Drosophila is an element located in several genes that regulate segmentation and segment identity; it has homologues in the genomes of vertebrate species and a number of homoeo box-containing recombinant DNA clones have been isolated from Xenopus, man and mouse. In the present study we have isolated from a library of murine DNA a recombinant lambda clone (H24.1) which contains a sequence closely homologous to the homoeo box within the Drosophila Antennapedia (Antp) gene. The protein sequence of the homoeo domain is identical to that encoded by Hu-1, one of a the pair of closely linked homoeo boxes in the human genome. We have used a sensitive RNase protection assay to examine transcription of the H24.1 gene during mouse development, and in adult tissues. We report that the gene is transcribed from as early as 7.5 days post coitum (p.c.), with maximum expression at days 11.5 and 12.5 p.c. The transcript is enriched in embryonic spinal cord and brain at day 12.5 p.c., and in adult kidney.
Human chromosome 17 contains a cluster of at least three distinct homeo box regions, Hu1, Hu2, and Hu5, within a 20 kb stretch of DNA. A mouse homeo box region, Mu1, which maps to chromosome 11, was isolated and found to contain extensive nucleotide homology with a 4 kb region encompassing the Hu1 homeo box. The conservation of these chromosomal regions between man and mouse was confirmed by nucleotide sequence analysis: approximately 380 bp of DNA are more than 90% homologous and the 61 amino acids of the homeo box domain are perfectly conserved. We found that a human teratocarcinoma cell line expresses high levels of Hu1 homeo-box-containing mRNA only after differentiation of the cells following treatment with retinoic acid. In contrast, mouse teratocarcinoma cells did not express Mu1 homeo-box-containing mRNA at any stage of differentiation, whereas the expression of such transcripts was detected in mouse embryos from 10 to 17 days of gestation.
A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described. The method depends upon annealing poly(adenylic acid)-rich mRNA to oligothymidylic acid-cellulose columns and its elution with buffers of low ionic strength. Biologically active rabbit globin mRNA has been purified by this procedure and assayed for its ability to direct the synthesis of rabbit globin in a cell-free extract of ascites tumor. Inasmuch as various mammalian mRNAs appear to be rich in poly(adenylic acid) and can likely be translated in the ascites cell-free extract, this approach should prove generally useful as an initial step in the isolation of specific mRNAs.
Identification of cells has been made in stained smears of cell suspensions prepared from mouse testes and separated by velocity sedimentation at unit gravity. Comparison of various methods of producing suspensions demonstrated that the best cell separations were achieved using suspensions prepared with trypsin. Various fractions obtained following separation contained 29% Sertoli cells sedimenting at about 14 mm/h, 17% Leydig cells at 11 mm/h, 73% pachytene spermatocytes at 9.5 mm/h, 54% binucleate spermatids and 14% secondary spermatocytes at 6.7 mm/h, 77% round spermatids at 4.5 mm/h, 21% elongating spermatids and 74% cytoplasmic fragments detached from these spermatids at 2.1 mm/h and 37% late spermatids at 0.75 mm/h. The resolution of different size classes of cells was essentially complete, but separation of different types of cells was limited by the occurrence of multinucleate forms of the cells and by fragments of damaged elongated spermatids. Most cells, however, appeared to be intact on light microscopical examination.
An autosomally inherited condition is described in the mouse which causes genetic females to develop as phenotypic males. XX males are phenotypically normal with the exception of small testes, which, in the adult, are devoid of germ cells. During late fetal and early postnatal development, male-type germ cells are present but progressively become lost, so that none is present by 10 days of age. XO males are also phenotypically normal, but spermatogenesis is active in the testis, and spermatozoa are produced. Cytological evidence of a Y-autosome translocation was completely lacking, and it is proposed that an autosomal dominant gene mutation, analogous to that found in other mammalian species, is responsible for the sex reversal.Copyright © 1971 S. Karger AG, Basel
The condition of testicular feminization in mice resembles that in man and other mammals. It is clearly X-linked. There is no evidence at present that the gene for testicular feminization is involved in mouse spermatogenesis, at least up to the spermatocyte stage.
Chicken embryo tissues were examined for the expression of pp60c-src, the normal cellular homolog of the transforming protein
of Rous sarcoma virus. Three assays, including a solid-phase radioimmunoassay, a competitive radioimmunoprecipitation assay,
and an immune complex protein kinase assay, were employed. Elevated levels of pp60c-src were detected in lysates from several
neural tissues, including brain, retina, and spinal ganglia. Other tissues contained 8- to 10-fold-lower levels of pp60c-src,
levels comparable to those found in chicken embryo fibroblasts. Expression of pp60c-src in brain tissues was also shown to
vary with the developmental stage of the embryo.
We have localized normal cellular pp60c-src in the developing chick neural retina by immunocytochemical staining using antisera raised against bacterially expressed pp60v-src, the src gene product of Rous sarcoma virus. pp60c-src was expressed in developing retinal neurons at the onset of differentiation. Expression of pp60c-src persisted in mature neuronal cells that were postmitotic, fully differentiated, and functional. pp60c-src immunoreactivity was localized within processes and cell bodies of ganglion neurons, processes of rods and cones, and in some but not all neurons of the inner nuclear layer. Protein kinase assays and Western transfer analyses identified the immunoreactive protein as pp60c-src, and confirmed that its expression occurs at the time the first neuronal cells in the retina differentiate. We conclude from these studies that pp60c-src is the product of a developmentally regulated gene that is more important in neuronal differentiation or function than cell proliferation.
A differential hybridization screening procedure has identified cDNAs which correspond to RNAs which are expressed in mouse
testis and at lower levels in liver and spleen. The sensitivity of this procedure is such that approximately 0.5% of 1.4 ×
104 cDNA clones are revealed as “testis specific”. We have focused on ten CDNA clones which have been used to identify RNAs expressed
in the haploid phase of spermatogenesis. Using Northern blots to analyse RNA isolated from the testes of mutant mice (Tfm/Y and Sxr/+) blocked at specific stages in spermatogenesis or RNA from sexually immature mice, 8 clones have been identified which
correspond to RNAs expressed uniquely or at much higher levels in meiotic or post meiotic cells.
Cellular genes that are homologous to the transforming genes of certain RNA tumor viruses are suspected to play a functional role during normal developmental processes. To investigate this further, we are studying the expression of the cellular homolog of the Rous sarcoma virus transforming gene (c-src) during embryogenesis of fish, frog, and chicken by quantitative determination of the activity of the c-src encoded protein kinase (pp60c-src). The kinase activity from embryos of fish, frog, and chicken displays the same enzymatic characteristics as the kinase from adult animals: It phosphorylates only tyrosine residues in protein substrates, and its activity is relatively insensitive to inhibition by the diadenosine nucleotide Ap4A. During the course of development, the varying kinase activity level reflects differential expression of the c-src gene product. The kinase activity is low during early development, increases dramatically during organogenesis, and decreases thereafter to the level found in adult animals. The kinase activity displays an organ specificity, with brain showing the highest activity in embryos as well as in adults. Muscle, however, shows high activities during organogenesis, but no or barely detectable activity in adult animals. Our data suggest, therefore, that the c-src gene product plays more of a role in differentiation than in proliferation processes during embryogenesis, and that it may act as a pleiotropic effector.
We have asked whether oncogenesis by the mouse mammary tumor virus (MMTV), a slowly oncogenic retrovirus, involves integration of viral DNA within a certain region of the host genome. We first identified a C3H mouse mammary tumor bearing a single new MMTV provirus and cloned a 19 kilobase (kb) DNA restriction fragment containing a junction of viral and host sequences. Host sequences from this clone were used to retrieve 25 kb of the uninterrupted locus (termed MMTV int1) from a bacteriophage library of normal mouse DNA. Hybridization with subcloned DNA fragments of MMTV int1 detected abnormal restriction fragments in digests of DNA from 18 of 26 C3H mammary tumors. The rearrangements all appeared to be due to the insertion of an MMTV provirus, and the integration sites were located in at least five clusters over a total distance of 19 kb. A polyadenylated 2.6 kb RNA species transcribed from int1 was found in the few tumors tested, but not in lactating mammary glands from C3H mice. Of 12 tested viral oncogenes, none exhibited homology with cloned DNA from this locus. We propose that tumorigenesis by MMTV is strongly favored by proviral insertion within the int1 locus, perhaps as a consequence of enhanced expression of a novel cellular oncogene.
Most mammary carcinomas induced in C3H mice by the mouse mammary tumour virus (MMTV) bear a new proviral insertion within a highly conserved locus on chromosome 15 called int-1. A transcriptional unit within this locus is inactive in all tested normal tissues but expressed at low levels in mammary tumours with proviral insertions positioned on either the 5' and 3' sides of the gene. Transcription of the proviruses proceeds away from int-1; thus an indirect mechanism appears to activate expression of this putative oncogene.
Evidence is mounting that cellular oncogenes are involved in tumorigenesis, whatever its proximal cause. Some of these genes may serve to initiate the genesis of tumors, some to sustain the final neoplastic phenotype, some may serve either purpose in different contexts. Cellular oncogenes appear not to be tumorigenic in their native state; they must either be activated to abnormal levels of expression, or mutated so as to change some aspect of their function. Based on present evidence, either of these mechanisms may apply. The recognition and isolation of cellular oncogenes has set the stage for fresh assaults on the mechanisms of carcinogenesis. The role of DNA damage can now be examined directly and the nature of the damage explored. The number of genetic functions required for the genesis of an individual tumor may become clear. The genes responsible for heritable predispositions to cancer can be sought. We hope to discern the variety and nature of the biochemical functions that can drive a cell to cancerous growth. The design of definitive strategies for the prevention or cure of human malignancy may have to await all of these accomplishments.
The normal cellular protein pp60c-src is a tyrosine-specific protein kinase that is homologous to the transforming protein of Rous sarcoma virus (RSV) but its function is unknown. The expression of pp60c-src in chick and human embryonic tissues was monitored by the immune complex protein kinase assay, Western transfer analysis, and immunocytochemical staining at the light microscope level. pp60c-src kinase was expressed in the head and trunk regions of the chick embryo at all stages of development examined; however, expression increased significantly during the major period of organogenesis (Hamburger and Hamilton stages 21 to 32). Western transfer analysis showed that the amount of pp60c-src protein increased in parallel with the increase in kinase activity. Highest levels of pp60c-src kinase were present in the neural tube, brain, and heart of the stage 32 chick embryo. Lower levels of activity were found in eye, limb bud, and liver. Immunocytochemical staining of the neural tube region and heart of the chick confirmed the results of biochemical analysis and showed immunoreactive pp60c-src distributed throughout the neural tube and heart. The distribution of pp60c-src kinase in human fetal tissues was similar to that in the chick embryo; elevated levels of pp60c-src kinase were present in cerebral cortex, spinal cord, and heart, but all other tissues examined expressed some pp60c-src kinase. The results of our studies suggest that pp60c-src plays a fundamental role in an aspect of cellular metabolism that is particularly important in electrogenic tissues.
In somatic cells the level of myc transcription is not restricted to particular cell types but correlates closely with the rate of cell division. Such transcription involves the use of two active myc promoters and produces two messenger RNA species that are differentially represented among the transcripts of different tissues. In apparent contrast to somatic cells, mitotically and meiotically dividing germ cells have very few myc transcripts and appear to proliferate, at least for a few divisions, in the absence of myc transcription. These results raise interesting questions regarding the role of the myc gene product in terminally differentiating cells, particularly of the germ line series.