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Normal insulin secretion requires the coordinated functioning of [beta]-cells within pancreatic islets. This coordination depends on a communications network that involves the interaction of [beta]-cells with extracellular signals and neighboring cells. In particular, adjacent [beta]-cells are coupled via channels made of connexin36 (Cx36). To assess the function of this protein, we investigated islets of transgenic mice in which the Cx36 gene was disrupted by homologous recombination. We observed that compared with wild-type and heterozygous littermates that expressed Cx36 and behaved as nontransgenic controls, mice homozygous for the Cx36 deletion (Cx3[6.sup.-/-]) featured [beta]-cells devoid of gap junctions and failing to exchange microinjected Lucifer yellow. During glucose stimulation, islets of Cx3[6.sup.-/-] mice did not display the regular oscillations of intracellular calcium concentrations ([[Ca.sup.2+].sub.i]) seen in controls due to the loss of cell-to-cell synchronization of [[Ca.sup.2+].sub.i] changes. The same islets did not release insulin in a pulsatile fashion, even though the overall output of the hormone in response to glucose stimulation was normal. However, under nonstimulatory conditions, islets lacking Cx36 showed increased basal release of insulin. These data show that Cx36-dependent signaling is essential for the proper functioning of [beta]-cells, particularly for the pulsatility of [[Ca.sup.2+].sub.i] and insulin secretion during glucose stimulation., Normal insulin secretion requires the coordinated functioning of [beta]-cells within pancreatic islets. This coordination is achieved through extracellular signals and ligands of the connective matrix as well as through the [...]

Gap junctional intercellular communication (GJIC) has been suggested to be involved in early embryonic development but the actual functional role remained elusive. Connexin (Cx) 43 and Cx45 are co‐expressed in embryonic stem (ES) cells, form gap junctions and are considered to exhibit adhesive function and/or to contribute to the establishment of defined communication compartments. Here, we describe the generation of Cx43/Cx45‐double deficient mouse ES cells to achieve almost complete breakdown of GJIC. Cre‐loxP induced deletion of both, Cx43 and Cx45, results in a block of differentiation in embryoid bodies (EBs) without affecting pluripotency marker expression and proliferation in ES cells. We demonstrate that GJIC‐incompetent ES cells fail to form primitive endoderm in EB cultures, representing the inductive key step of further differentiation events. Lentiviral overexpression of either Cx43 or Cx45 in Cx43/45 mutants rescued the observed phenotype, confirming the specificity and indicating a partially redundant function of both connexins. Upon differentiation GJIC‐incompetent ES cells exhibit a strikingly altered subcellular localization pattern of the transcription factor NFATc3. Control EBs exhibit significantly more activated NFATc3 in cellular nuclei than mutant EBs suggesting that Cx‐mediated communication is needed for synchronized NFAT activation to induce orchestrated primitive endoderm formation. Moreover, pharmacological inhibition of NFATc3 activation by Cyclosporin A, a well‐described inhibitor of calcineurin, phenocopies the loss of GJIC in control cells. StemCells2017;35:859–871 Our study set out to identify the functional role of gap junctional intercellular communication (GJIC) in ES cells. The deletion of Connexin43 and Connexin45 leads to an abrogation of GJIC, which resulted in defective primitive endoderm formation in embryoid bodies (EBs). We assume that IP3 diffuses via gap junction conduits to coordinate and sustain NFATc3 activation in a synchronized manner. As a consequence of Cx43/45 deletion the IP3 mediated synchronization breaks down and the development of primitive endoderm is impaired. A similar phenotype is observed upon blocking NFAT signaling using the calcineurin inhibitor Cyclosporin A. The fluorescence pictures show EBs on day6 of differentiation. The primitive endoderm marker Gata6 is shown in red, the pluripotency marker Nanog in green. Scale: 20 µm.

The keratitis-ichthyosis-deafness (KID) syndrome is characterized by corneal, skin, and hearing abnormalities. KID has been linked to heterozygous dominant missense mutations in the GJB2 and GJB6 genes, encoding connexin26 and 30, respectively. In vitro evidence indicates that KID mutations lead to hyperactive (open) hemichannels, which in some cases is accompanied by abnormal function of gap junction channels. Transgenic mouse models expressing connexin26 KID mutations reproduce human phenotypes and present impaired epidermal calcium homeostasis and abnormal lipid composition of the stratum corneum affecting the water barrier. Here we have compiled relevant data regarding the KID syndrome and propose a mechanism for the epidermal aspects of the disease.

Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS)2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.

Sudden infant death syndrome (SIDS) describes the sudden, unexplained death of a baby during its first year of age and is the third leading cause of infant mortality. It is assumed that 20 of all SIDS cases are because of cardiac arrhythmias resulting from mutations in ion channel proteins. Besides ion channels also cardiac gap junction channels are important for proper conduction of cardiac electric activation. In the mammalian heart Connexin43 (Cx43) is the major gap junction protein expressed in ventricular cardiomyocytes. Recently, a novel Connexin43 loss-of-function mutation (Cx43E42K) was identified in a 2-month-old SIDS victim.

Mammalian kidneys are rich in sulfatides. Papillary sulfatides, especially, contribute to renal adaptation to chronic metabolic acidosis. Due to differences in their cer­amide (Cer) anchors, the structural diversity of renal sulfatides is large. However, the underling biological function of this complexity is not understood. As a compound's function and its tissue location are intimately connected, we analyzed individual renal sulfatide distributions of control and Cer synthase 2 (CerS)2-deficient mice by imaging MS (IMS) and by LC-MS2(in controls for the cortex, medulla, and papillae separately). To explain locally different structures, we compared our lipid data with regional mRNA levels of corresponding anabolic enzymes. The combination of IMS and in source decay-LC-MS2analyses revealed exclusive expression of C20-sphingosine-containing sulfatides within the renal papillae, whereas conventional C18-sphingosine-containing compounds were predominant in the medulla, and sulfatides with a C18-phytosphingosine were restricted to special cortical structures. CerS2 deletion resulted in bulk loss of sulfatides with C23/C24-acyl chains, but did not lead to decreased urinary pH, as previously observed in sulfatide-depleted kidneys. The reasons may be the almost unchanged C22-sulfatide levels and constant total renal sulfatide levels due to compensation with C16- to C20-acyl chain-containing compounds. Intriguingly, CerS2-deficient kidneys were completely depleted of phytosphingosine-containing cortical sulfatides without any compensation.

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre+;Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre+;Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.

Gap junctional coupling of renin-producing cells is of major functional importance for the control of renin synthesis and release. This study was designed to determine the relevance of the vascular gap junction protein connexin 45 (Cx45) for the control of renin expression and secretion. By crossbreeding mice which drive Cre recombinase under the control of the endogenous renin promoter with mice harboring floxed Cx45 gene alleles, we generated viable mice with a deletion of Cx45 in the renin cell lineage. These mice were normotensive, and renin cells in their kidneys were normal with regard to localization and number. Sodium deficiency induced typical recruitment of renin-producing cells along afferent arterioles, whereas sodium overload resulted in a decrease in the number of cells expressing renin. Regulation of renin secretion by perfusion pressure, catecholamines, and angiotensin II from isolated kidneys of mice with renin cell-specific deletion of Cx45 was normal. Analyzing Cx45 promoter activity in cells of the preglomerular arteriolar tree by using mice driving the reporter gene LacZ under the control of the Cx45 promoter revealed strong staining in smooth muscle cells of the media, whereas renin-expressing cells were almost devoid of LacZ staining. Conversely, renin-producing cells, but not vascular smooth muscle cells expressed the gap junction protein Cx40. These findings suggest that Cx45 plays no major functional role in renin-producing cells, probably because the expression of Cx45 is downregulated in these cells. Since renin-producing cells in the adult kidney can reversibly transform into vascular smooth muscle cells and vice versa, our findings on connexin expression indicate that these phenotype switches are paralleled by characteristic reciprocal changes in the transcriptional activity of Cx40 and Cx45 genes.

The predominant testicular gap junctional protein connexin43 (cx43) is located between neighboring Sertoli cells (SCs) and between SCs and germ cells. It is assumed to be involved in testicular development, cell differentiation, initiation, and maintenance of spermatogenesis with alterations of its expression being correlated with various testicular disorders. Because total disruption of the cx43 gene leads to perinatal death, we generated a conditional cx43 knockout (KO) mouse using the Cre/loxP recombination system, which lacks the cx43 gene solely in SCs (SCCx43KO), to evaluate the SC-specific functions of cx43 on spermatogenesis in vivo. Adult SCCx43KO−/−mice showed normal testis descent and development of the urogenital tract, but testis size and weight were drastically lower compared with heterozygous and wild-type littermates. Histological analysis and quantitation of mRNA expression of germ cell-specific marker genes revealed a significant reduction in the number of spermatogonia but increased SC numbers/tubule with only a few tubules left showing normal spermatogenesis. Thus, SC-specific deletion of cx43 mostly resulted in an arrest of spermatogenesis at the level of spermatogonia or SC-only syndrome and in intratubular SC clusters. Our data demonstrate for the first time that cx43 expression in SCs is an absolute requirement for normal testicular development and spermatogenesis.

Connexins (Cxs) 40, 43, and 45 are expressed in many different tissues, but most abundantly in the heart, blood vessels, and the nervous system. We examined formation and gating properties of heterotypic gap junction (GJ) channels assembled between cells expressing wild-type Cx40, Cx43, or Cx45 and their fusion forms tagged with color variants of green fluorescent protein. We show that these Cxs, with exception of Cxs 40 and 43, are compatible to form functional heterotypic GJ channels. Cx40 and Cx43 hemichannels are unable or effectively impaired in their ability to dock and/or assemble into junctional plaques. When cells expressing Cx45 contacted those expressing Cx40 or Cx43 they readily formed junctional plaques with cell-cell coupling characterized by asymmetric junctional conductance dependence on transjunctional voltage, Vj. Cx40/Cx45 heterotypic GJ channels preferentially exhibit Vj-dependent gating transitions between open and residual states with a conductance of ∼42pS; transitions between fully open and closed states with conductance of ∼52pS in magnitude occur at substantially lower (∼10-fold) frequency. Cx40/Cx45 junctions demonstrate electrical signal transfer asymmetry that can be modulated between unidirectional and bidirectional by small changes in the difference between holding potentials of the coupled cells. Furthermore, both fast and slow gating mechanisms of Cx40 exhibit a negative gating polarity.

More than 97% of mice in which the C-terminal region of connexin43 (Cx43) was removed (designated as Cx43K258stop) die shortly after birth due to a defect of the epidermal barrier. The abnormal expression of Cx43K258stop protein in the uppermost layers of the epidermis seems to perturb terminal differentiation of keratinocytes. In contrast to Cx43-deficient mice, neonatal Cx43K258stop hearts show no lethal obstruction of the right ventricular outflow tract, but signs of dilatation. Electrocardiographies of neonatal hearts reveal repolarization abnormalities in 20% of homozygous Cx43K258stop animals. The very rare adult Cx43K258stop mice show a compensation of the epidermal barrier defect but persisting impairment of cardiac function in echocardiography. Female Cx43K258stop mice are infertile due to impaired folliculogenesis. Our results indicate that the C-terminally truncated Cx43K258stop mice lack essential functions of Cx43, although the truncated Cx43 protein can form open gap junctional channels.

Astrocytes secrete cytokines and neurotrophic factors to neurons, consistent with a neurosupportive role for astrocytes. However, in ischemic or metabolic insults, the function of astrocytic gap junctions composed mainly from connexin43 (Cx43) remains controversial. We have previously shown that heterozygous Cx43 null mice subjected to middle cerebral artery occlusion exhibited significantly enhanced stroke volume and apoptosis compared to wild-type mice. In this study, we used mice in which the human GFAP promoter-driven cretransgene deletes the floxed Cx43gene in astrocytes, excluding the effects from reduced Cx43 expression in many other cell types as well as astrocytes. We induced focal brain ischemia in mice lacking Cx43 in astrocytes [Cre(+)] and control littermates [Cre(−)]. Cre(+) mice showed a significantly increased stroke volume and enhanced apoptosis, detected by terminal dUTP nick-end labeling and caspase-3 immunostaining, compared to Cre(−) mice. Inflammatory response assessed by the microglial marker CD11b was amplified in the penumbra of Cre(+) mice compared to that of Cre(−) mice. Our results suggest that astrocytic gap junctions could be important for the regulation of neuronal apoptosis and the inflammatory response after stroke. These findings support the view that astrocytes play a critical role in neuroprotection during ischemic insults.

Recent evidence suggests that electrotonic coupling is an important mechanism for neuronal synchronisation in the mammalian cortex and hippocampus. Various types of network oscillations have been shown to depend on, or be sharpened by, gap junctions between inhibitory interneurones or excitatory projection cells. Here we made use of a targeted disruption of the gene coding for Cx36, a recently discovered neuronal gap junction subunit, to analyse its role in hippocampal network behaviour. Mice lacking Cx36 are viable and lack obvious morphological or behavioural abnormalities. Stimulation of afferent and efferent fibre pathways in hippocampal slices revealed a largely normal function of the synaptic circuitry, including tetanically evoked network oscillations. Spontaneous sharp waves and ripple (∼200 Hz) oscillations, however, occurred less frequently in slices from Cx36 ‐/‐ mice, and ripples were slightly slower than in littermate controls. Moreover, epileptiform discharges elicited by 4‐aminopyridine were attenuated in slices from Cx36 ‐/‐ mice. Our findings indicate that Cx36 plays a role in the generation of certain forms of network synchronisation in the hippocampus, namely sharp wave‐ripple complexes and hypersynchronous epileptiform discharges.

AbstractGap junctions are clustered channels between contacting cells through which direct intercellular communication via diffusion of ions and metabolites can occur. Two hemichannels, each built up of six connexin protein subunits in the plasma membrane of adjacent cells, can dock to each other to form conduits between cells. We have recently screened mouse and human genomic data bases and have found 19 connexin (Cx) genes in the mouse genome and 20 connexin genes in the human genome. One mouse connexin gene and two human connexin genes do not appear to have orthologs in the other genome. With three exceptions, the characterized connexin genes comprise two exons whereby the complete reading frame is located on the second exon. Targeted ablation of eleven mouse connexin genes revealed basic insights into the functional diversity of the connexin gene family. In addition, the phenotypes of human genetic disorders caused by mutated connexin genes further complement our understanding of connexin functions in the human organism. In this review we compare currently identified connexin genes in both the mouse and human genome and discuss the functions of gap junctions deduced from targeted mouse mutants and human genetic disorders.

Gap junctions are composed of protein subunits, called connexins, and provide a pathway for the exchange of ions and small molecules between contacting cells. This transfer of molecules is thought to be an important pathway for direct cell communication, and is involved in tissue homeostasis, growth control and embryonic development. Impairment of gap junctional intercellular communication (GJIC) via different mechanisms may therefore contribute to dysregulated cellular proliferation and subsequent tumor development. We investigated the effect of Connexin32-deficiency on liver histology and the formation of preneoplastic foci and hepatocellular neoplasms in transgenic knockout mice, as Connexin32 (Cx32) is the major gap junction protein in the liver. Loss of Cx32 does not alter the morphology of extrafocal liver tissue. However, after administration of a single dose of diethylnitrosamine (DEN), given 2 weeks after birth, the number and volume fraction of preneoplastic foci showed a 3.3-fold to 12.8-fold increase in the Cx32-deficient mice as compared with the corresponding wildtype groups, regardless of sex and age of the animals. Number and volume fraction of hepatocellular adenomas and carcinomas also increased significantly in these animals. The experimental groups did not differ in the morphology of the different types of preneoplastic foci and neoplasms. On the other hand, Cx32-deficiency without DEN treatment did not lead to an increase in the spontaneous development of any type of preneoplastic hepatic foci or hepatocellular neoplasms in up to 18-month-old Cx32-deficient mice as compared with wildtype controls. In conclusion, our results indicate that impairment of GJIC in mouse liver due to deletion of the Cx32 coding DNA clearly promotes the carcinogenic effect of DEN administration and results in a higher susceptibility to hepatocellular neoplasms, but does not appear to initiate hepatic tumor development.

Communication and interaction between cells has been shown to be important during the embryonic development of the vertebrate hindbrain, which becomes transiently subdivided into segments called rhombomeres (r). One gene family allowing intercellular communication and possibly being involved in the control of hindbrain development is the connexin family encoding gap junction channels. Here, we have characterized in detail the previously observed (Dahl et al., 1997) expression of one particular connexin gene, connexin31 (Cx31), in the mouse embryonic hindbrain and compared it with that of Cx43 and Cx36. We found transient Cx31 expression from approximately embryonic day (E) E8–E11 in two small lateral/dorsal subgroups of cells in the hindbrain. We could show that these spots of expression corresponded to r3 and r5 and that Cx31 expression in r3 and r5 was controlled by the transcription factor Krox20. In contrast, expression of Cx43 and Cx36 started later (from E9.5 and E10.5, respectively) and was confined to longitudinal stripes of expression. In addition, from E10.5–E11.5, Cx31 was expressed by a column of cells in ventral r4, most likely representing contralateral vestibulo-acoustic efferent neurons, immediately anterior to a ventral column expressing Cx36 at the same stage. From E11.5 onward, another site of Cx31 expression was detected in the boundary cap cells in the entry/exit points of all mixed sensory/motor and in the entry points of pure sensory nerves. This expression was not present in the boundary cap cells of the exit points of pure motor nerves. So far, our analysis of the hindbrain area of Cx31-deficient embryos in terms of projections of sensory or motor neurons or in the generation or migration of neurons has not yet revealed any obvious defects. © 2002 Wiley-Liss, Inc.

AbstractA novel mouse gap junction gene, coding for a presumptive protein of 258 amino acids (molecular mass: 28981 Da), has been designated connexin29. This single copy gene was mapped to distal mouse chromosome 5 and shows 75% sequence identity to a human connexin30.2 sequence in the database. Connexin29 mRNA (4.4 kb) is highly expressed in mouse sciatic nerve and less abundant in spinal cord as well as in adult brain, where it increased 12-fold between day 7 and 14 post partum. Our expression data suggest that the new connexin gene is active in myelinforming glial cells.

The rodent gap junction protein connexin32 (Cx32) is highly expressed in hepatocytes, less abundantly in Schwann cells and oligodendrocytes, and at low levels in the early mouse embryo. In both hepatocytes and Schwann cells, Cx32 expression is directed by alternative promoter regions (P1 and P2) which activate differently spliced transcript isoforms. Here we describe a third Cx32 transcript isoform expressed in embryonic cells, oocytes, and liver. Using competitive polymerase chain reaction, we have found that this new Cx32 transcript containing exon 1A is 200-fold less abundant in liver than the Cx32 isoform with exon 1. In mouse oocytes, the exon 1A-containing Cx32 transcript is exclusively expressed. Immunoblot analyses revealed no Cx32 protein expression in embryonic stem cells, whereas it has previously been demonstrated in oocytes. When the putative Cx32 promoter region upstream of exon 1A was cloned before the lacZreporter gene, transient transfection yielded weak expression in embryonic stem cells. Our results suggest that the exon 1A-containing Cx32 isoform is likely to be inherited as an oogenetic product but not translated during early embryogenesis.

Mutations in the human GJB3gene that codes for Connexin31 (Cx31), a protein subunit of gap junction channels, have recently been reported to cause deafness and the skin disorder erythrokeratodermia variabilis.To study the function of this gene in mice, we generated animals with targeted replacement of the Cx31 gene (Gjb3) by a lacZreporter gene. Although homozygous Cx31-deficient adult mice (Gjb3−/−) were found among the offspring of heterozygous Cx31-deficient parents (Gjb3+/−), 60% of the animals expected according to Mendelian inheritance were lost between ED 10.5 and 13.5. Placentas of Gjb3−/−embryos at ED 9.5 were smaller than controls as a result of severely reduced labyrinth and spongiotrophoblast size. From ED 10.5 onward, placentas of surviving Gjb3−/−embryos recovered progressively and reached normal size and morphology by ED 18.5. This corresponds to a time period in which another connexin isoform, Connexin43, is upregulated in spongiotrophoblast cells of Cx31-deficient and control placentas. No morphological or functional defects of skin or inner ear were observed in surviving adult Gjb3−/−mice. We conclude that Cx31 is essential for early placentation but can be compensated for by other connexins in the embryo proper and adult mouse.

Using the Cre/loxP system, we have circumvented early postnatal lethality and possible pleiotropic effects of general Cx43 gene deletion, in order to determine the expression and function of connexin43 (Cx43) in defined cell types. General or cell type-specific, Cre-mediated deletion of the floxed (i.e. flanked by loxP sites) Cx43-coding region led to activation of the inserted lacZ reporter gene in cells with transcriptional activity of the Cx43 gene. As deduced from lacZ expression in mice with general deletion, transcriptional activity of the Cx43 gene was not only found in a broad range of cell types known to a express Cx43, but also in pancreatic duct cells and vascular cells of the gut and skeletal muscle. Cre-mediated deletion restricted to defined cell types led to lacZ activation highlighting corresponding subsets of cells expressing Cx43, such as vascular endothelial cells, hepatic duct cells and putative neural crest cells, which were otherwise masked by strong Cx43 expression in neighbouring cells. In Cx43 expressing cell types, the floxed Cx43 allele was useful as a Cre-excision reporter for the characterization of Cre transgenes.

In order to reveal the biological function(s) of the gap-junction protein connexin 45 (Cx45), we generated Cx45-deficient mice with targeted replacement of the Cx45-coding region with the lacZ reporter gene. Heterozygous Cx45+/− mice showed strong expression of the reporter gene in vascular and visceral smooth muscle cells. Cx45-deficient embryos exhibited striking abnormalities in vascular development and died between embryonic day (E) 9.5 and 10.5. Differentiation and positioning of endothelial cells appeared to be normal, but subsequent development of blood vessels revealed impaired formation of vascular trees in the yolk sac, impaired allantoic mesenchymal ingrowth and capillary formation in the labyrinthine part of the placenta, and arrest of arterial growth, including a failure to develop a smooth muscle layer surrounding the major arteries of the embryo proper. As a consequence, the hearts of most Cx45-deficient embryos were dilated. The abnormal development of the vasculature in the yolk sac of Cx45−/− embryos could be caused by defective TGFβ signalling, as the amount of TGF β1 protein in the epithelial layer of the yolk sac was largely decreased in the E9.5 Cx45−/− embryo, compared with the wild-type embryo. The defective vascular development was accompanied by massive apoptosis, which began in some embryos at E8.5 and was abundant in virtually all tissues of the embryos at E9.5. We conclude that in Cx45−/− embryos, vasculogenesis was normal, but subsequent transformation into mature vessels was interrupted. Development of different types of vessels was impaired to a varying extent, which possibly reflects the complementation by other connexin(s).

To analyze the molecular basis of gap junctional communication in mouse retina, we examined the expression pattern of the following 13 connexin (Cx) genes: Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx46, and Cx50. By using reverse transcriptase‐polymerase chain reactions with primer oligonucleotides to murine connexin genes, we detected mRNAs of Cx26, Cx31, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, and Cx50. Retinae from heterozygous mice with targeted replacement of most of the Cx45 open reading frame by a lacZ reporter gene showed Cx45 promoter activity in somata of the ganglion cell layer and the inner nuclear layer. Immunoblot and immunofluorescence analyses with antibodies generated to murine connexin epitopes revealed the presence of Cx36, Cx37, Cx43, and Cx45 proteins: The outer and inner plexiform layer were immunopositive for Cx36 and Cx45. Cx37 immunoreactivity was found in blood vessels of the inner retina. Cx43 immunolabeling was detected in the ganglion cell layer and nerve fiber layer where it was largely colocalized with immunostaining of glial fibrillary acidic protein suggesting that Cx43‐positive cells could be of glial origin. No Cx26 protein was detected in retina by using Cx26 antibodies for immunoblot analyses or confocal microscopy. Furthermore, comparative immunofluorescence analyses of retinae from mice deficient for Cx31, Cx32, or Cx40 with retinae of wild‐type mice revealed no specific immunostaining. Our results demonstrate regional specificity in expression of connexin genes in mouse retina and, thus, provide a basis for future assignments of functional defects in connexin‐deficient mice to cells in different regions of the retina. J. Comp. Neurol. 425:193–201, 2000. © 2000 Wiley‐Liss, Inc.

Intercellular propagation of signals through connexin32- containing gap junctions is of major importance in physiological processes like nerve activity-dependent glucose mobilization in liver parenchymal cells and enzyme secretion from pancreatic acinar cells. In these cells, as in other organs, more than one type of connexin is expressed. We hypothesized that different permeabilities towards second messenger molecules could be one of the reasons for connexin diversity. In order to investigate this, we analyzed transmission of inositol 1,4,5-trisphosphate-mediated calcium waves in FURA-2-loaded monolayers of human HeLa cells expressing murine connexin26, -32 or -43. Gap junction-mediated cell coupling in different connexin- transfected HeLa cells was standardized by measuring the spreading of microinjected Mn2+ that led to local quenching of FURA-2 fluorescence. Microinjection of inositol 1,4,5-trisphosphate into confluently growing HeLa connexin32 transfectants induced propagation of a Ca2+ wave from the injected cell to neighboring cells that was at least three- to fourfold more efficient than in HeLa Cx26 cells and about 2.5-fold more efficient than in HeLa Cx43 transfectants. Our results support the notion that diffusion of inositol 1,4,5-trisphosphate through connexin32- containing gap junctions is essential for the optimal physiological response, for example by recruiting liver parenchymal cells that contain subthreshold levels of this short lived second messenger.

Here we describe several gene targeting approaches currently used in our laboratory for the generation of deletion or replacement mutants of connexin genes in the mouse and discuss the advantage of the double-replacement strategy for the generation of conditional mutants. For the analysis of complementary functions of connexins, it will be necessary to generate mice with mutations in several connexin genes. We also report how this can be effectively accomplished. The replacement of targeted connexin-coding DNA with a reporter gene, to mimic expression of the deleted gene product, is currently being used in several laboratories. The use of different reporter genes or their differently localized gene products could allow distinction of promoter activity in double or triple connexin mutant mice.

Affinity-purified antibodies to oligopeptides derived from two different regions of the carboxyterminus and cytoplasmic loop or to the last 103 C-terminal amino acids of mouse connexin37 (Cx37) were used to characterize expression of this gap junctional protein in endothelium of several murine tissues. Cx37 was expressed in endothelium of large blood vessels in brain, liver, kidney, spleen, heart, and lung, but not in capillaries. In addition, weak Cx37 immuno-signals were observed in lung respiratory epithelium of small bronchi and in alveolar epithelial cells of bronchioli. The ratios of Cx37 protein to Cx37 mRNA in adult and embryonic kidney as well as skin were 29–303-fold larger than in lung, suggesting that Cx37 mRNA was translated at different efficiencies in kidney and skin versus lung. Cx37 protein was more abundant in embryonic kidney and lung than in the corresponding adult tissues. After differential centrifugation of plasma membrane fractions in sucrose gradients, we found that Cx37-containing gap junctions in lung were much smaller than Cx32 and Cx26 aggregates from liver. HeLa cells were transfected with mouse Cx37 cDNA. In these cells, mouse Cx37 protein was phosphorylated mainly at serine, less at tyrosine, and very little at threonine residues. Three conductance states were resolved at 110, 240, and 315 pS.

Abstract:  Hepatocytes were isolated from wild-type and connexin32-deficient (Cx32-deficient) mice. Pairs of cells were chosen to study the electrical properties of gap junction channels using the dual voltage-clamp method. The total gap junction currents revealed that Cx32-deficient hepatocytes express one type of connexin (Cx26) and wild-type hepatocytes express two types of connexins (Cx26 and Cx32). The unitary gap junction currents suggest that Cx32-deficient cells have homotypic channels (Cx26–Cx26) while wild-type cells form homotypic (Cx26–Cx26, Cx32–Cx32) and heterotypic channels (Cx26–Cx32). Homotypic channels exhibited a main conductance and a residual conductance, both virtually insensitive to gap junction voltage (V j) (Cx32–Cx32: γj,main=31 pS, γj,residual=9 pS; Cx26–Cx26: γj,main=102 pS, γj,residual=17 pS). Residual states were regularly seen in Cx32–Cx32 channels, but rarely in Cx26–Cx26 channels. Heterotypic channels showed a main conductance and a residual conductance. The former was sensitive to V j (average γj,main=52 pS). The electrophysiological data suggest that Cx32 hemichannels are more abundant than Cx26 hemichannels in prenatal (ratio 4:1) and adult wild-type hepatocytes (ratio 23:1) and that the total number of gap junction channels is larger in prenatal cells than in adult cells. The diversity of the relationship g j,ss/g j,inst=f(V j) (g j,ss: gap junction conductance at steady state; g j,inst: instantaneous gap junction conductance; V j: transjunctional voltage) seen in wild-type cells suggests that the ratio Cx26/Cx32 hemichannels is variable among hepatocytes. A comparison of total and unitary conductances implies that Cx26 hemichannels are down-regulated in Cx32-deficient cells and that docking between Cx26 and Cx32 hemichannels occurs randomly. While the gap junction currents are compatible with homotypic and heterotypic channels, the presence of heteromeric channels cannot be excluded.

1Human HeLa cells expressing mouse connexin30 (Cx30) were used to study the electrical properties of Cx30 gap junction channels. Experiments were performed on cell pairs with the dual voltage‐clamp method.2The gap junction conductance (gj) at steady state showed a bell‐shaped dependence on junctional voltage (Vj; Boltzmann fit: Vj,0= 27 mV, gj,min= 0.15, z= 4). The instantaneous gjdecreased slightly with increasing Vj.3The gap junction currents (Ij) declined with time following a single exponential. The time constants of Ijinactivation (τi) decreased with increasing Vj.4Single channels exhibited a main state, a residual state and a closed state. The conductances γj,mainand γj,residualwere 179 and 48 pS, respectively (pipette solution, potassium aspartate; temperature, 36‐37 °C; extrapolated to Vj= 0 mV).5The conductances γj,residualand γj,mainshowed a slight Vjdependence and were sensitive to temperature (Q10values of 1.28 and 1.16, respectively).6Current transitions between open states (i.e. main state, substates, residual state) were fast (< 2 ms), while those between an open state and the closed state were slow (12 ms).7The open channel probability (Po) at steady state decreased from 1 to 0 with increasing Vj(Boltzmann fit: Vj,0= 37 mV; z= 3).8Histograms of channel open times implied the presence of a single main state; histograms of channel closed times suggested the existence of two closed states (i.e. residual states).9We conclude that Cx30 channels are controlled by two types of gates, a fast one responsible for Vjgating involving transitions between open states (i.e. residual state, main state), and a slow one correlated with chemical gating involving transitions between the closed state and an open state.

A new mouse connexin gene has been isolated that codes for a connexin protein of 505 amino acid residues. Based on the predicted molecular mass of 57.115 kDa, it has been designated connexin-57. Similar to most other mouse connexin genes, the coding region of connexin-57 is not interrupted by introns and exists in the mouse genome as a single-copy gene. Within the connexin family, this new gene shows highest sequence identity to porcine connexin-60 in the α group of connexins. The connexin-57 gene was mapped to a position on mouse chromosome 4, 30 centimorgans proximal to a cluster of previously mapped connexin genes. Low levels of connexin-57 mRNA were detected in skin, heart, kidney, testis, ovary, intestine, and in the mouse embryo after 8 days post coitum, but expression was not detected in brain, sciatic nerve or liver. In order to analyze gene function, the connexin-57 coding region was expressed by transfection in human HeLa cells, where it restored homotypic intercellular transfer of microinjected neurobiotin. Heterotypic transfer was observed between HeLa connexin-57 transfectants and HeLa cells, expressing murine connexin-43, -37, or -30.3. Double whole-cell voltage clamp analyses revealed that HeLa-connexin-57 transfectants expressed about 10 times more channels than parental HeLa cells. Voltage gating by transjunctional and transmembrane voltages as well as unitary conductance (∼27 picosiemens) were different from intrinsic connexin channels in parental HeLa cells.

The connexins are a family of proteins that form the intercellular membrane channels of gap junctions. Genes encoding 13 different rodent connexins have been cloned and characterized to date. Connexins vary both in their distribution among adult cell types and in the properties of the channels that they form. In order to explore the functional significance of connexin diversity, several mouse connexin-encoding genes have been disrupted by homologous recombination in embryonic stem cells. Although those experiments have illuminated specific physiological roles for individual connexins, the results have also raised the possibility that connexins may functionally compensate for one another in cells where they are coexpressed. In the present study, we have tested this hypothesis by interbreeding mice carrying null mutations in the genes (Gjb1 and Gja1) encoding connexin32 (β1 connexin) and connexin43 (α1 connexin), respectively. We found that fetuses lacking both connexins survive to term but, as expected, the pups die soon thereafter from the cardiac abnormality caused by the absence of connexin43. A survey of the major organ systems of the doubly mutant fetuses, including the thyroid gland, developing teeth, and limbs where these two connexins are coexpressed, failed to reveal any morphological abnormalities not already seen in connexin43 deficient fetuses. Furthermore, the production of thyroxine by doubly mutant thyroids was confirmed by immunocytochemistry. We conclude that, at least as far as the prenatal period is concerned, the normal development of those three organs in fetuses lacking connexin43 cannot simply be explained by the additional presence of connexin32 and vice-versa. Either gap junctional coupling is dispensable in embryonic and fetal cells in which these two connexins are coexpressed, or coupling is provided by yet another connexin when both are absent. Dev. Genet. 24:5–12, 1999. © 1999 Wiley-Liss, Inc.

Connexins are subunits of gap junction channels, which mediate the direct transfer of ions, second messenger molecules and other metabolites between contacting cells. Gap junctions are thought to be involved in tissue homeostasis, embryonic development and the control of cell proliferation . It has also been suggested that the loss of intercellular communication via gap junctions may contribute to multistage carcinogenesis . We have previously shown that transgenic mice that lack connexin32 (Cx32), the major gap junction protein expressed in hepatocytes, express lower levels of a second hepatic gap junction protein, Cx26, suggesting that Cx32 has a stabilizing effect on Cx26 . Here, we report that male and female one-year-old mice deficient for Cx32 had 25-fold more and 8-fold more spontaneous liver tumors than wild-type mice, respectively. Incorporation of bromodeoxyuridine (BrdU) into the liver was higher for Cx32-deficient mice than for wild-type mice, suggesting that their hepatocyte proliferation rate was higher. Furthermore, intraperitoneal injection, two weeks after birth, of the carcinogen diethylnitrosamine (DEN) led, after one year, both to more liver tumors in Cx32-deficient mice than in controls, and to accelerated tumor growth. Loss of Cx32 protein from hepatic gap junctions is therefore likely to cause enhanced clonal survival and expansion of mutated (‘initiated’) cells, which results in a higher susceptibility to hepatic tumors. Our results demonstrate that functional gap junctions inhibit the development of spontaneous and chemically induced tumors in mouse liver.

A new gap junction gene isolated from rat brain cDNA, mouse retina cDNA and mouse genomic DNA is called connexin36, since it codes for a connexin protein of 321 amino acids corresponding to the theoretical molecular mass of 36 045 kDa (rat) and 36 084 kDa (mouse). Only one amino acid residue differs between rat and mouse connexin36. In the single murine connexin36 gene, an 1.14‐kb intron interrupts the coding region, similar as in the homologous skate connexin35 gene. Because of this unique feature, mouse connexin36 differs from the other 13 murine connexin genes and is suggested to form a new δ subclass of connexins. Connexin36 mRNA (2.9 kb) is highly expressed in adult retina and less abundant in brain where it gradually increased during fetal development until day 7 post partum, and decreased thereafter.

Intercellular communication is mediated by specialized cell-cell contact areas known as gap junctions. Connexins are the constitutive proteins of gap junction intercellular channels. Various cell expression systems are used to express connexins and, in turn, these expression systems can then be tested for their ability to form functional cell-cell channels. In this review, expression of murine endogenous connexins in primary cells and established cell lines is compared with results obtained by expression of exogenous connexins inXenopus oocytes and cultured mammalian cells. In addition, first reports on characterization of connexin-deficient mice are discussed.

By screening of a rat liver cDNA library with complex and deoxyinosine containing oligonucleotide probes a cDNA clone was isolated and shown by sequencing to code for the amino-terminal half of the rat liver 28 kDa gap junction protein. The insert hybridized to a 1.9 kb species from rat and mouse liver poly(A) +RNA in Northern blot analysis. In embryonic mouse hepatocytes the amount of the 1.9 kb mRNA increased 3-fold between 24 and 96 h in culture. This correlates with the previously described increase of the 28 kDa gap junction protein under these conditions.

The effect of heptanol, an alkanol which decreases gap junctional permeability, was investigated in the perfused rat pancreas. Under basal conditions, heptanol (3.5 μM) caused a three- to fourfold increase of pancreatic juice, protein, and amylase outputs. The effect on enzyme secretion was fully reversible upon removal of the alkanol and was not secondary to the release of acetylcholine from nerve endings, since it was not prevented by addition of atropine (10−6M) to the perfusate. By contrast, another alkanol, hexanol (3 μM), which does not decrease coupling between acinar cells in spite of anesthetic properties analogous to those of heptanol, did not alter pancreatic secretion. The effect of heptanol was not mediated by a significant stimulation of cyclic AMP, nor did the alkanol increase the secretion of lactic dehydrogenase, a cytosolic marker. Analysis of the numerical density of freeze-fractured and immunolabeled gap junctions between acinar cells did not show differences between heptanol-perfused and control pancreases. In addition, heptanol did not alter carbachol (10−6Mand 10−5M)-evoked amylase release. Since heptanol blocks cell coupling, apparently without interfering with the main intra-cellular pathways triggering enzyme release, we suggest that downregulation of direct cell-to-cell communications increases pancreatic exocrine secretion. Thus, cell coupling is probably involved in the regulation of the secretory activity of acinar cells.

We have isolated and characterized a human genomic clone containing the complete coding region of connexin31 (Cx31). Similar to rodent Cx31, the coding region of human Cx31 is completely contained within the second exon and consists of 810 nucleotides. The deduced human Cx31 polypeptide consists of 270 amino acids with a predicted molecular mass of 30.818 kDa. Its sequence is most similar to mouse Cx31 (82.6% identical amino acids) and rat (83.0% identical amino acids), but shows considerably fewer potential sites of phosphorylation. After Northern blot hybridization, two Cx31 transcripts of 2.2 and 1.8 kb were detected in total RNA of the human keratinocyte cell line HaCaT and two transcripts of 2.2 and 1.9 kb in total RNA of E6/E7 transfected human keratinocytes (HEK cells). Using affinity-purified rabbit antibodies to mouse Cx31, immunofluorescence analysis demonstrated relatively weak expression of human Cx31 in HaCaT and HEK cells. The Cx31 gene exists as a single copy gene in the human genome and was mapped to the chromosomal region 1p34–p36 by analyzing human–mouse somatic cell hybrids.

Bacillus subtiliscells grown in yeast extract medium accumulated 3-fluoro-l-erythro-[1,2-14C2]malate more than 30-fold from the surrounding medium. No metabolic products derived from 3-fluoro-l-erythro-malate could be detected in these cells. l-Malate competitively inhibited transport of 3-fluoro-l-erythro-malate. This malate analogue was itself a competitive inhibitor of l-malate uptake. Cells that had been grown in yeast extract supplemented with 5 mM l-malate showed a 10-fold increased affinity towards 3-fluoro-l-erythro-malate relative to cells grown in yeast extract medium with no added malate. Our results suggest that two transport systems for l-malate can be induced in B. subtilis. The first of these systems seems to effect uptake of C4-dicarboxylates (l-malate, succinate, and fumarate) in yeast extract medium. The second transport system (or possibly a modification of the first transport system) seems to be induced by addition of l-malate to this medium and is also functioning in malate minimal medium.

The connexin genes Cx31 and Cx45 coding for proteins of gap junctional subunits have been assigned to mouse chromosomes 4 and 11 by Southern blot hybridization of specific gene probes to DNA from mouse × Chinese hamster somatic cell hybrids. In addition, our results confirm the recent assignment of mouse connexin genes Cx26, Cx32, Cx37, Cx40, Cx43, and Cx46 to mouse chromosomes 14, X, 4, 3, 10, and 14, respectively, by analysis of interspecific backcrosses and by somatic cell hybridization. Our assignment of the Cx31 gene to mouse chromosome 4 locates the fourth connexin gene on this mouse chromosome to which the genes for Cx31.1, Cx37, and Cx30.3 have previously been assigned. Interestingly three of them (coding for Cx31, Cx31.1, and Cx30.3) are preferentially expressed in skin. Possibly some of the connexin genes clustered on mouse chromosome 4 may be regulated coordinately.

The chromosomal integration site of the structural gene of Moloney murine leukemia virus (M-MuLV) in the genome of BALB/Mo mice was mapped genetically. These mice transmit the exogenous M-MuLV as an endogenous virus at a single Mendelian locus. Two independent experimental approaches were used: (i) Non-virus-producing fibroblasts prepared from homozygous BALB/Mo embryos were fused to Chinese hamster Wg3-h-o cells. In an analysis of 30 independent mouse-Chinese hamster cell hybrid clones, the segregation of the viral genome measured by molecular hybridization and enzymes assigned to 16 different mouse chromosomes were compared. We found a highly concordant segregation of M-MuLV sequences and the mouse enzyme triosephosphate isomerase (TPI, EC 5.3.1.1), whose gene has been assigned to chromosome 6. A further karyotype analysis of 9 clones, in which the chromosomes were identified cytochemically, supported this result. (ii) The segregation of the viral genome was studied in backcrosses of BALB/Mo with ABP/J mice. In the backcross ABP/J×(ABP/J×BALB/Mo) a linkage of the M-MuLV genome to the morphological marker wa-1on mouse chromosome 6 was found. This confirmed the conclusion that the M-MuLV genome is integrated in mouse chromosome 6. These experiments define the genetic locus Mov-1, denoting the genetically transmitted structural gene of M-MuLV in BALB/Mo mice.

Under selective growth conditions a revertant of mouse cells, defective in hypoxanthine phosphoribosyltransferase activity (HPRT, EC-No. 2.4.2.8), was isolated, which contained an electrophoretically abnormal form of HPRT activity. The specific HPRT activity in crude extracts of the revertant cells is about 30% of the level determined in normal wild type cells. The variant HPRT reacts with antiserum against normal mouse HPRT but the rate of heat inactivation of the variant activity is different from the wild type form. By isozyme and karyotype analyses of somatic cell hybrids between the revertant mouse cells and Chinese hamster cells we found that the abnormal HPRT activity is coded for by the mouse X-chromosome as expected for a mutation in the structural HPRT gene.

Changes in the total amount of the gap junction protein (Mr26,000) after partial hepatectomy or bile duct ligation and recanalization were investigated in rat liver membranes by quantitative immunoblot with rabbit antiserum to the Mr26,000 protein. The loss and reappearance of the Mr26,000 protein roughly paralleled loss and reappearance of gap junction plaques analyzed previously under similar physiological conditions by freeze-fracture of hepatocyte surfaces. The total amount of the hepatic Mr26,000 protein in liver plasma membranes and the total area of the hepatocyte surface occupied by gap junction plaques appeared to be proportional under these conditions. However, at the minimum, 28-35 hr after partial hepatectomy we still find about 15% of the Mr26,000 protein, in contrast to <1% of gap junction plaques, determined by morphometric analysis. This discrepancy is probably due to the fact that very small gap junction plaques, single connexons, and free Mr26,000 gap junction subunits are missed by the morphometric analysis. At the times of the minimal amount of the Mr26,000 protein in hepatic plasma membranes after partial hepatectomy or bile duct ligation we found that crude hepatic lysosomal membranes of these rats contained less Mr26,000 protein than lysosomal membranes of nonoperated control animals. Thus, we conclude that the decrease and increase of the total amount of the Mr26,000 protein cannot be explained only by dispersal and reuse of gap junction subunits but are largely due to degradation and resynthesis of the Mr26,000 protein. No significant change in the amount of the Mr21,000 protein that had been isolated with gap junction plaques was observed in liver plasma membranes after partial hepatectomy. This confirms our previous conclusion that the Mr26,000 and Mr21,000 proteins are independent of each other.

If a chromosomal fragment transferred into recipient cells were integrated or strongly associated with a specific recipient chromosome it should segregate with this chromosome in hybrid cells. In order to corroborate this prediction we studied two independent mouse cell clones (“transferent clones”) which had taken up by chromosomal gene transfer a human chromosomal fragment carrying the gene for cytosol thymidine kinase (TKs, E.C.No.2.7.1.75). The following results were obtained.1.Ten somatic cell hybrids isolated after fusion of transferent mouse clones and chinese hamster cells expressed functional human TKs and mouse galactokinase (GALK, E.C.No.2.7.1.6) activity. Counterselected derivatives of all clones had lost human TKs but still expressed mouse GALK. Recently both mouse genes for TKs and GALK have been assigned to mouse chromosome 11 (Kozak and Ruddle, 1977a, McBreen et al., 1977). Therefore our results argue against integration or association of the human gene for TKs at the site of the homologous defective mouse TKs-GALK-region in the genome of transferent clones.2.In three somatic cell hybrids isolated after fusion of microcells from two different transferent mouse clones with established chinese hamster cells only human TKs but not mouse GALK was expressed. Karyotypic analysis of one hybrid suggested the presence of at least one copy of mouse chromosome 9 per hybrid cell. Chromosome 9 and the mouse isozyme activity of mannose phosphate isomerase which had been previously assigned to a gene locus on mouse chromosome 9 were missing in a subclone counter-selected against the presence of TKs activity. These findings suggested that the transferred human gene for TKs may be integrated or strongly associated with mouse chromosome 9 in this transferent mouse clone.3.Two other somatic cell hybrids were analyzed which were derived from a different phenotypically stable transferent mouse clone by similar microcell fusion with the chinese hamster cells. Although these hybrids expressed human TKs activity, no mouse chromosome could be detected in these clones. Possibly after fusion of microcells derived from certain transferent mouse clones the transferred human chromosomal fragment could become translocated from a mouse chromosome to the chinese hamster genome.

Four independent mouse gene transfer clones, i.e. mouse cells which had taken up a human chromosomal fragment carrying the human gene for hypoxanthine phosphoribosyltransferase (HPRT), were characterized with the following results:1.None of the clones expressed human a-galactosidase activity whose structural gene has been previously located on the human X-chromosome in the neighbourhood of the gene coding for HPRT. Thus the human chromosomal fragment in these mouse cells is presumably so small that it does not carry the gene for human a-galactosidase A.2.All the gene transfer clones when maintained in logarithmic growth for at least 100 generations in selective medium became phenotypically stable, i.e. they no longer segregated the transferred human HPRT activity after being shifted to non-selective growth conditions.3.Somatic cell hybrids were isolated after fusion of phenotypically stable mouse gene transfer cells, expressing human HPRT, with chinese hamster cells, mouse cells which were derived from the same parental line as the gene transfer cells but which expressed reverted mouse HPRT were also fused with these chinese hamster cells. All isolated hybrid cell clones were counter-selected for the absence of functional HPRT.

An established Chinese hamster cell line was fused with microcells isolated from phenotypically stable transferent mouse cells which contained a mouse transgenome coding for an abnormal form of mouse hypoxanthine phosphoribosyltransferase (HPRT, EC. No. 2.4.2.8) (Willecke et al. 1979). Two hybrids were isolated which expressed the abnormal form of mouse HPRT but no mouse a-galactosidase (GALA, EC. No. 3.2.1.22). In one of these microcell hybrids the abnormal HPRT activity segregated under counter-selective conditions with mouse chromosome 3. No mouse chromosome or additional mouse gene marker was found in the second microcell hybrid, possibly because of breakage and/or rearrangement of the integrated transgenome during the isolation of this hybrid. We conclude from these results that the transferred mouse HPRT gene in a phenotypically stable clone is not integrated at its homologous site on the host X chromosome. Rather, the transgenome is probably integrated into mouse chromosome 3, possibly due to homologies in repeated DNA sequences which may occur in the transgenome and which are interspersed at many sites in the host genome.

The 12 known connexin genes coding for the subunit proteins of the gap junction channels and related in nucleotide sequence are widely dispersed in the mouse genome. By using a series of mouse × Chinese hamster somatic cell hybrids and molecular probes of murine connexin genes, we have assigned the connexin 50 gene to mouse chromosome 3. The connexin 33 gene has been mapped to the X chromosome, thus confirming the previous chromosomal assignment of this gene based on interspecific back-cross mapping.

Inhibition of de novo fatty acid biosynthesis by the antibiotic cerulenin in Bacillus subtilisstopped de novo synthesis of neutral lipids and phospholipids. The bacteria ceased growing but remained completely viable. Addition of 12-methyltetradecanoic acid and palmitic acid to the culture medium of cerulenin-treated cells restored growth of the bacteria, albeit at a reduced rate. Although the de novo synthesis of all lipid components of the membrane was blocked, citrate-Mg2+transport activity remained inducible, and induced cells did not lose this transport activity when treated with cerulenin. Shortly after the addition of cerulenin, the rate of ribonucleic acid synthesis dropped rapidly and was followed by a slower decrease in the rate of protein synthesis. The rate of deoxyribonucleic acid synthesis remained almost unaffected. The rapid decrease of ribonucleic acid synthesis in cerulenin-treated cells might be due to the inhibition of de novo fatty acid biosynthesis or it might be due to a secondary effect of cerulenin in B. subtiliscells.

We have characterized the pattern of connexin expression in embryonic and extraembryonic tissues during early mouse development. In the preimplantation blastocyst, at 3.5 days post coitum (dpc), immunofluorescent signals specific for connexin31 and connexin43 proteins were present in both the inner cell mass and the trophectoderm, as shown by confocal laser scan microscopy. Immediately after implantation at 6.5 dpc, however, we find complete compartmentation of these two connexins: connexin31 mRNA and protein are expressed exclusively in cells derived from the trophectoderm lineage, whereas connexin43 mRNA and protein are detected in cells derived from the inner cell mass. This expression pattern of connexin31 and connexin43 is maintained at 7.5 dpc when the axial polarity of the mouse embryo is established. It correlates with the communication compartments in extraembryonic tissues and the gastrulating mouse embryo, respectively. The communication boundary between those compartments may be due to incompatibility of connexin31 and connexin43 hemichannels, which do not communicate with each other in cell culture.

Citrate transport can be induced in a glycerol-requiring mutant of Bacillus subtiliseven after deprival of glycerol. Under these conditions de novo phospholipid synthesis is stopped although ribonucleic acid, deoxyribonucleic acid, and protein syntheses continue. It is concluded that the inducibility of bacterial transport systems must not necessarily require de novo phospholipid synthesis.

The uptake of several dicarboxylic acids by Bacillus subtilishas been surveyed. Gratuitous conditions for induction of l-malate uptake were achieved under which the addition of l-malate to the medium resulted in increased uptake of l-malate by the cells without significantly influencing the growth rate.