Oocytes

An extracellular serine protease (Ac1) with a molecular mass of 35 kDa was puriWed from the nematodetrapping fungus Arthrobotrys conoides. The optimum activity of Ac1 is at pH 7.0 and 53.2°C (over 20 min). Ac1 can degrade a broad range of substrates including casein, gelatin, bovine serum albumin, collagen, and nematode cuticles. Moreover, the enzyme can immobilize the freeliving nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, indicating Ac1 may be involved in infection against nematodes. The encoding gene of Ac1 contains one intron of 60-bp and two exons encoding a polypeptide of 411 amino acid residues. The deduced polypeptide sequence of Ac1 showed a high degree of similarity to two previously reported serine proteases PII and Mlx from other nematode-trapping fungi (81% aa sequence identity). However, three proteases Ac1, Aoz1 and Mlx showed optimum temperatures at 53.2, 45 and 65°C, respectively. Compared to PII, Ac1 appears to have a signiWcantly higher activity against gelatin, bovine serum albumin, and non-denatured collagen. Moreover, our bioassay experiments showed that Ac1 is more eVective at immobilizing P. redivivus than B. xylophilus.

Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii. ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii, were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H ؉ and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracellular H ؉. ChR2 is expressed mainly in C. reinhardtii under low-light conditions, suggesting involvement in photoreception in dark-adapted cells. The predicted seventransmembrane ␣ helices of ChR2 are characteristic for G proteincoupled receptors but reflect a different motif for a cation-selective ion channel. Finally, we demonstrate that ChR2 may be used to depolarize small or large cells, simply by illumination. voltage clamp ͉ patch clamp ͉ light sensitive ͉ Chlamydomonas reinhardtii ͉ Xenopus laevis oocyte P hotoreception in animals evolved differently in vertebrates and invertebrates although both use as primary photoreceptor rhodopsin, a seven-transmembrane (7-TM) helix protein with covalently linked retinal. In vertebrates, light-activated rhodopsin initiates a G protein-coupled enzyme cascade that ends in the hydrolysis of cGMP and the closure of the cGMP-regulated cation channels, thus hyperpolarizing the plasma membrane (1-3). Invertebrates also use a rhodopsin-based G proteincoupled signaling cascade, which, however, results in activation of transient receptor potential (TRP)͞TRP-like (TRPL)channels and a depolarization of the membrane potential (4, 5). Microbial-type rhodopsins are also 7-TM retinal proteins but show no sequence homology to animal rhodopsins. The prototype microbial-type rhodopsin is the light-driven proton pump bacteriorhodopsin (BR) (6), a structurally and functionally well understood model system for active transmembrane ion transport (7, 8). Other microbial rhodopsins are light-driven chloride pumps or light sensors, the latter enabling phototaxis by coupling to specific transducers (9-11). Recently, microbial-type rhodopsins were also found in fungi (12) and algae (13), and it has been known since 1991 that phototaxis and photophobic responses in the green alga Chlamydomonas reinhardtii are mediated by rhodopsins with a microbial-type all-trans retinal chromophore (14-16). As shown for the green algae Haematococcus pluvialis (17), C. reinhardtii (18), and Volvox carteri (19), the photoreceptor currents are confined to the pigmented eyespot region. Although the ion dependence of the photoreceptor currents in these algae is different, it has been proposed for all three species on the basis of stimulus-response

into an FFF instrument (Model F-1000-FO, FFFractionation; LLC, Salt Lake City, UT ) operating with 0.007% Triton X-100 in water mobile phase at 2 ml min Ϫ1 and a cross-flow rate of 0.5 ml min Ϫ1 . 17. Electrodeposition was performed by placing 20 l of the nanotube suspension on the surface of a freshly cleaved HOPG substrate (Advanced Ceramics, Cleveland, OH), confining the droplet within a viton O-ring (4-mm outer diameter, 1.7 mm thick), capping the trapped suspension with a stainless steel electrode on top of the O-ring, and applying a steady voltage of 1.1 V for 6 min. When suspended in water, the nanotubes are negatively charged and are therefore driven by the electric field onto the HOPG surface. After deposition, the HOPG-nanotube surface was washed with methanol on a spin coater to remove the water and Triton X-100 surfactant.

Complementary sets of genes are epigenetically silenced in male and female gametes in a process termed genomic imprinting. The Dnmt3L gene is expressed during gametogenesis at stages where genomic imprints are established. Targeted disruption of Dnmt3L caused azoospermia in homozygous males, and heterozygous progeny of homozygous females died before midgestation. Bisulfite genomic sequencing of DNA from oocytes and embryos showed that removal of Dnmt3L prevented methylation of sequences that are normally maternally methylated. The defect was specific to imprinted regions, and global genome methylation levels were not affected. Lack of maternal methylation imprints in heterozygous embryos derived from homozygous mutant oocytes caused biallelic expression of genes that are normally expressed only from the allele of paternal origin. The key catalytic motifs characteristic of DNA cytosine methyltransferases have been lost from Dnmt3L, and the protein is more likely to act as a regulator of imprint establishment than as a DNA methyltransferase.

The M-current regulates the subthreshold electrical excitability of many neurons, determining their firing properties and responsiveness to synaptic input. To date, however, the genes that encode subunits of this important channel have not been identified. The biophysical properties, sensitivity to pharmacological blockade, and expression pattern of the KCNQ2 and KCNQ3 potassium channels were determined. It is concluded that both these subunits contribute to the native M-current. The M-current is a slowly activating and deactivating potassium conductance that plays a critical role in determining the subthreshold electrical excitability of neurons as well as the responsiveness to synaptic inputs (1-3). The M-current was first described in peripheral sympathetic neurons (4, 5), and differential expression of this conductance produces subtypes of sympathetic neurons with distinct firing patterns (3). The M-current is also expressed in many neurons in the central nervous system (CNS) (1, 6, 7). To date, the molecular identity of the channels underlying the M-current remains unknown. Here we show that the KCNQ2 and KCNQ3 channel subunits can coassemble to form a channel with essentially identical biophysical properties and pharmacological sensitivities to the native M-current and that the pattern of KCNQ2 and KCNQ3 gene expression is consistent with these genes encoding the native M-current. The KCNQ potassium channel gene family has three members: KCNQ1 (KvLQT1), KCNQ2, and KCNQ3 (8-12). Injection of KCNQ2 mRNA into Xenopus oocytes resulted in the consistent expression of a relatively small potassium current that is slowly activating and deactivating (Fig. 1A). The properties of this channel are essentially identical to those described previously (11). In contrast, injection of KCNQ3 mRNA did not result in the expression of a current above background level. When the KCNQ2 and KCNQ3 mRNAs were coinjected, however, the resultant current was 11-fold larger than

RNA interference (RNAi) is a mechanism by which doublestranded RNAs (dsRNAs) suppress specific transcripts in a sequence-dependent manner. dsRNAs are processed by Dicer to 21-24-nucleotide small interfering RNAs (siRNAs) and then incorporated into the argonaute (Ago) proteins 1-4 . Gene regulation by endogenous siRNAs has been observed only in organisms possessing RNA-dependent RNA polymerase (RdRP) . In mammals, where no RdRP activity has been found, biogenesis and function of endogenous siRNAs remain largely unknown. Here we show, using mouse oocytes, that endogenous siRNAs are derived from naturally occurring dsRNAs and have roles in the regulation of gene expression. By means of deep sequencing, we identify a large number of both 25-27-nucleotide Piwi-interacting RNAs (piRNAs) and 21-nucleotide siRNAs corresponding to messenger RNAs or retrotransposons in growing oocytes. piRNAs are bound to Mili and have a role in the regulation of retrotransposons. siRNAs are exclusively mapped to retrotransposons or other genomic regions that produce transcripts capable of forming dsRNA structures. Inverted repeat structures, bidirectional transcription and antisense transcripts from various loci are sources of the dsRNAs. Some precursor transcripts of siRNAs are derived from expressed pseudogenes, indicating that one role of pseudogenes is to adjust the level of the founding source mRNA through RNAi. Loss of Dicer or Ago2 results in decreased levels of siRNAs and increased levels of retrotransposon and protein-coding transcripts complementary to the siRNAs. Thus, the RNAi pathway regulates both protein-coding transcripts and retrotransposons in mouse oocytes. Our results reveal a role for endogenous siRNAs in mammalian oocytes and show that organisms lacking RdRP activity can produce functional endogenous siRNAs from naturally occurring dsRNAs.

The subunit stoichiometry of the mammalian K+ channel KVl .l (RCKl) was examined by linking together the coding sequences of 2-5 K+ channel subunits in a single open reading frame and tagging the expression of individual subunits with a mutation (Y379K or Y379R) that altered the sensitivity of the channel to block by external tetraethylammonium ion. Two lines of evidence argue that these constructs lead to K+ channel expression only through the formation of functional tetramers. First, currents expressed by tetrameric constructs containing a single mutant subunit have a sensitivity to tetraethylammonium that is well fitted by a single site binding isotherm. Second, a mutant subunit fY379K) that expresses only as part of a heteromultimer contributes to the expression of functional channels when coexpressed with a trimeric construct but not a tetrameric construct.

Xenopus oocytes convert a continuously variable stimulus, the concentration of the maturation-inducing hormone progesterone, into an all-or-none biological response-oocyte maturation. Here evidence is presented that the all-or-none character of the response is generated by the mitogen-activated protein kinase (MAPK) cascade. Analysis of individual oocytes showed that the response of MAPK to progesterone or Mos was equivalent to that of a cooperative enzyme with a Hill coefficient of at least 35, more than 10 times the Hill coefficient for the binding of oxygen to hemoglobin. The response can be accounted for by the intrinsic ultrasensitivity of the oocyte's MAPK cascade and a positive feedback loop in which the cascade is embedded. These findings provide a biochemical rationale for the all-or-none character of this cell fate switch.

DNA methylation is essential for the control of a number of biological mechanisms in mammals [1]. Mammalian development is accompanied by two major waves of genome-wide demethylation and remethylation: one during germ-cell development and the other after fertilisation [2-7]. Most previous studies have suggested that the genome-wide demethylation observed after fertilisation occurs passively, that is, by the lack of maintenance methylation following DNA replication and cell division [6,7], although one other study has reported that replication-independent demethylation may also occur during early embryogenesis [8]. Here, we report that genes that are highly methylated in sperm are rapidly demethylated in the zygote only hours after fertilisation, before the first round of DNA replication commences. By contrast, the oocyte-derived maternal alleles are unaffected by this reprogramming. They either remain methylated after fertilisation or become further methylated de novo. These results provide the first direct evidence for active demethylation of single-copy genes in the mammalian zygote and, moreover, reveal a striking asymmetry in epigenetic methylation reprogramming. Whereas paternally (sperm)-derived sequences are exposed to putative active demethylases in the oocyte cytoplasm, maternally (oocyte)-derived sequences are protected from this reaction. These results, whose generality is supported by findings of Mayer et al. , have important implications for the establishment of biparental genetic totipotency after fertilisation, the establishment and maintenance of genomic imprinting, and the reprogramming of somatic cells during cloning.

Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in <0.1 microl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation.

Neuropathic pain may be produced, at least in part, by the increased activity of primary afferent neurons. Studies have suggested that an accumulation of voltage-gated sodium channels at the site of peripheral nerve injury is a primary precursory event for subsequent afferent hyperexcitability. In this study, a human sodium channel (hPN3, SCN10A) has been cloned from the lumbar 4/5 dorsal root ganglia (DRG). Expression of hPN3 in Xenopus oocytes showed that this clone is a functional voltage-gated sodium channel. The amino acid sequence of hPN3 is most closely related to the rat PN3/SNS sodium channels which are expressed primarily in the small neurons of rat DRGs. The homologous relationship between rPN3 and hPN3 is defined by (i) a high level of sequence identity (ii) sodium currents that are highly resistant to tetrodotoxin (TTX) (iii) similar tissue distribution profiles and (iv) orthologous chromosomal map positions. Since rPN3/SNS has been implicated in nociceptive transmission, hPN3 may prove to be a valuable target for therapeutic agents against neuropathic pain. © 1998 International Association for the Study of Pain. Published by Elsevier Science B.V.

In addition, VEGF is a potent inducer of vascular perme-Farmington, Connecticut 06030-3501 ability, a prerequisite initial event for plasma exudation and fibrin clot formation, a matrix permissive for angiogenesis (Iruela-Arispe and Dvorak, 1997). However, fac-Summary tors and mechanisms that regulate morphogenesis of endothelial cells into capillary-like networks are poorly Vascular endothelial cells undergo morphogenesis understood. into capillary networks in response to angiogenic fac-

Parasites (clones 3D7, HB3 and K1) were cultured and inhibitory constants for drugs assayed as described 2 . The effect of the interaction between two antimalarial agents on the growth of P. falciparum in culture was assessed using isobole analysis, as described 30 .

Tri-methylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging and heterochromatin formation. Here we show that HP1a, -b, and -g are released from chromatin during the M phase of the cell cycle, even though tri-methylation levels of histone H3 lysine 9 remain unchanged. However, the additional, transient modification of histone H3 by phosphorylation of serine 10 next to the more stable methyl-lysine 9 mark is sufficient to eject HP1 proteins from their binding sites. Inhibition or depletion of the mitotic kinase Aurora B, which phosphorylates serine 10 on histone H3, causes retention of HP1 proteins on mitotic chromosomes, suggesting that H3 serine 10 phosphorylation is necessary for the dissociation of HP1 from chromatin in M phase. These findings establish a regulatory mechanism of protein-protein interactions, through a combinatorial readout of two adjacent post-translational modifications: a stable methylation and a dynamic phosphorylation mark.

Cyclic nucleotides modulate potassium (K) channel activity in many cells and are thought to act indirectly by inducing channel protein phosphorylation. Herein we report the isolation from rabbit of a gene encoding a K channel (Kcnl) that is specifically activated by cGMP and not by cAMP. Analysis of the deduced amino acid sequence (725 amino acids) indicates that, in addition to a core region that is highly homologous to Shaker K channels, Kcnl also contains a cysteine-rich region similar to that of ligand-gated ion channels and a cyclic nucleotide-binding region. Northern blot analysis detects gene expression in kidney, aorta, and brain. Kcnl represents a class of K channels that may be specifically regulated by cGMP and could play an important role in mediating the effects of substances, such as nitric oxide, that increase intracellular cGMP.

Since the number of ovarian follicles declines with increasing age, AMH levels might be used as a marker for ovarian ageing. Therefore, we studied the relationship between AMH levels and ovarian response during ovarian stimulation for IVF. METHODS: A total of 130 patients undergoing their first IVF treatment cycle using a long protocol with GnRH agonist was prospectively included. Blood withdrawal was performed and the number of antral follicles was assessed by ultrasound on day 3 of a spontaneous cycle. Poor response and the number of oocytes were used as primary outcome measures. In a random subset of 23 patients a GnRH agonist stimulation test was performed to investigate whether a rise in FSH and LH would affect AMH levels. RESULTS: The data of 119 patients were analysed. Serum AMH levels were highly correlated with the number of antral follicles (r ⍧ 0.77; P < 0.01) and the number of oocytes retrieved (r ⍧ 0.57, P < 0.01). A negative association was found between AMH levels and poor ovarian response (fewer than 4 oocytes or cycle cancellation; OR 0.82, 95% CI 0.75-0.90, P < 0.01). Inclusion of inhibin B and FSH concentrations to AMH in a multivariate model improved the prediction of ovarian response. The post GnRH agonist rise in FSH and LH levels did not influence AMH values. CONCLUSIONS: Poor response in IVF, indicative of a diminished ovarian reserve, is associated with reduced baseline serum AMH concentrations. In line with recent observations it appears that AMH can be used as a marker for ovarian ageing.

THE adenosine-5&#x27;-triphosphate (ATP) molecule is an extracellular messenger in neural and non-neural tissues, where it activates several cell-surface-receptor subtypes, including G-protein-coupled receptors and ligand-gated ion channels 1 . ATP-gated channels ...

Andersen's syndrome is characterized by periodic paralysis, cardiac arrhythmias, and dysmorphic features. We have mapped an Andersen's locus to chromosome tified in unrelated patients. Expression of two of these mutations in Xenopus oocytes revealed loss of funcdevelopmental signaling in addition to its previously recognized function in controlling cell excitability in Salt Lake City, Utah 84112 5 Neuromuscular Pathology skeletal muscle and heart.

Canalicular secretion of bile salts is a vital function of the vertebrate liver, yet the molecular identity of the involved ATP-dependent carrier protein has not been elucidated. We cloned the full-length cDNA of the sister of P-glycoprotein (spgp; M r ϳ160,000) of rat liver and demonstrated that it functions as an ATP-dependent bile salt transporter in cRNA injected Xenopus laevis oocytes and in vesicles isolated from transfected Sf9 cells. The latter demonstrated a 5-fold stimulation of ATP-dependent taurocholate transport as compared with controls. This spgp-mediated taurocholate transport was stimulated solely by ATP, was inhibited by vanadate, and exhibited saturability with increasing concentrations of taurocholate (K m Ӎ 5 M). Furthermore, spgp-mediated transport rates of various bile salts followed the same order of magnitude as ATP-dependent transport in canalicular rat liver plasma membrane vesicles, i.e. taurochenodeoxycholate > tauroursodeoxycholate ‫؍‬ taurocholate > glycocholate ‫؍‬ cholate. Tissue distribution assessed by Northern blotting revealed predominant, if not exclusive, expression of spgp in the liver, where it was further localized to the canalicular microvilli and to subcanalicular vesicles of the hepatocytes by in situ immunofluorescence and immunogold labeling studies. These results indicate that the sister of P-glycoprotein is the major canalicular bile salt export pump of mammalian liver.

In insects, each olfactory sensory neuron expresses between one and three ligand-binding members of the olfactory receptor (OR) gene family, along with the highly conserved and broadly expressed Or83b co-receptor. The functional insect OR consists of a heteromeric complex of unknown stoichiometry but comprising at least one variable odorant-binding subunit and one constant Or83b family subunit. Insect ORs lack homology to G-protein-coupled chemosensory receptors in vertebrates and possess a distinct seven-transmembrane topology with the amino terminus located intracellularly. Here we provide evidence that heteromeric insect ORs comprise a new class of ligand-activated non-selective cation channels. Heterologous cells expressing silkmoth, fruitfly or mosquito heteromeric OR complexes showed extracellular Ca2+ influx and cation-non-selective ion conductance on stimulation with odorant. Odour-evoked OR currents are independent of known G-protein-coupled second messenger pathways. The fast response kinetics and OR-subunit-dependent K+ ion selectivity of the insect OR complex support the hypothesis that the complex between OR and Or83b itself confers channel activity. Direct evidence for odorant-gated channels was obtained by outside-out patch-clamp recording of Xenopus oocyte and HEK293T cell membranes expressing insect OR complexes. The ligand-gated ion channel formed by an insect OR complex seems to be the basis for a unique strategy that insects have acquired to respond to the olfactory environment.

25. The specific DNA fragments leading to a lethal or growth-defective event after induction were amplified by PCR with plasmid-specific primers tetRfor1399 (5Ј-CAATACAATGTAGGCTGC-3Ј) and catUrev (5Ј-AGT-TCAT T TGATATGCCTCC-3Ј), and PCR SUPERMix (GiBCO-BRL life technologies). All PCRs were performed in a Perkin-Elmer 9600 thermal cycler. After addition of template DNA, the PCR mixture was denatured at 94°C for 2 min, followed by the thermal cycle (30 s at 94°C, 30 s at 51°C, and 1.5 min at 72°C). 26. DNA sequences of all antisense segments contained in strains listed in tables are available upon request, and identification numbers of unknown genes can be found on Web table 2 (24). 27. S. F. Altschul et al., Nucleic Acids Res. 25, 3389 (1997). 28. Supported in part by the U.S. Defense Advanced Research Projects Agency (N65236-97-1-5810). We thank A. Marra and D. McDevitt for helpful discussions, and reviewers for comments on the manuscript.

A DNA sequence encoding a G-proteincoupled receptor was isolated from a mouse genomic library. The predicted protein is similar in structure to the thrombin receptor and has a similar activation mechanism. When expressed in Xenopus laevis oocytes, the receptor was activated by low concentrations of trypsin (EC 3.4.21.4) and by a peptide (SLIGRL) derived from the receptor sequence, but was not activated by thrombin (EC 3.4.21.5). Trypsin failed to activate a mutant receptor in which the presumed cleavage site Arg-34-Ser-35 was changed to an Arg-Pro sequence. The agonist peptide (SLIGRL) activated equally well mutant and wild-type

Newcastle upon Tyne England cDNA and genomic clones encoding a7, a novel neuronal nicotinic acetylcholine receptor (nAChR) a subunit, were isolated and sequenced. The mature a7 protein (479 residues) has moderate homology with all other a and non-a nAChR subunits and probably assumes the same transmembrane topology. a7 transcripts transiently accumulate in the developing optic tectum between E5 and E16. They are present in both the deep and the superficial layers of El2 tectum. In Xenopus oocytes, the c17 protein assembles into a homo-oligomeric channel responding to acetylcholine and nicotine. The CJ channel desensitizes very rapidly, rectifies strongly above -20 mV, and is blocked by a-bungarotoxin.

DFNB (X-linked, autosomal dominant, and autosomal recessive, respectively). In general, autosomal recessive deafness has an early onset and is very severe. Autosomal dominant deafness, by contrast, more often develops slowly over several decades. It is hoped that genes * Zentrum fü r Molekulare Neurobiologie Hamburg identified in families with dominant deafness may also Universitä t Hamburg underlie some forms of presbyacusis. Martinistrasse 85 A large number of loci for nonsyndromic deafness D-20246 Hamburg were identified in the last 4 years. There are at least 19 Germany loci for autosomal dominant deafness (DFNA1 to DFNA19) † Institut Pasteur and 22 loci for DFNB. Sometimes, depending on the Unité de Gé né tique des Dé ficits Sensoriels particular mutation, the same gene can be involved in CNRS URA 1968 dominant or recessive deafness (Kelsell et al., 1997; Liu 25 Rue du Dr. Roux et al., 1997; Weil et al., 1997; Denoyelle et al., 1998; F-75724 Paris Cedex 15 Verhoeven et al., 1998; Mustapha et al., 1999). Several

Vacular endothelal growth factor (VEGF), also known as vascular permeability factor, induces endothelal proliferation in vitro and vscular permeability in Wivo. The human tranmembrane c-fns-like tyrosine kinase Fit-i has recently been identified as a VEGF receptor. Fit-i kinase has seven Immunoglobulin-like extraceilular domains and a kinas insert sequence, features shared by two other human geneencoded proteins, kinase insert domain-containing receptor (KDR) and FLT-4. In this study we show that the mouse homologue of KDR, Flk-1, Is a second functional VEGF receptor. Flk-1 binds VEGF with hi affnity, undergoes autophosphorylation, and mediates VEGF-dependent Ca2+ efflux in Xenopus oocytes lhjected with Flk-1 mRNA. We also demonstrate by in situ hybridization that Flk-1 protein expression In the mouse embryo is restricted to the vascular endothellum and the umbilical cord stroma. VEGF and Its receptors Flk-l/KDR and Flt-i may play a role in vascular development and regulation of vascular permeability.

To target the mouse Cdk2 locus, we knocked-in two loxP sites flanking coding exons 2 and 3 of Cdk2 by homologous recombination in ES cells . Removal of these sequences by a Cre recombinase eliminates the PSTAIRE cyclin-binding domain. Moreover, splicing from We targeted the locus encoding the cyclin-dependent kinase 2 (CDK2) by homologous recombination in mouse embryonic stem (ES) cells. Embryonic fibroblasts lacking CDK2 proliferate normally and become immortal after continuous passage in culture. Elimination of a conditional Cdk2 allele in immortal cells does not have a significant effect on proliferation. Cdk2 -/mice are viable and survive for up to two years, indicating that CDK2 is also dispensable for proliferation and survival of most cell types. But CDK2 is essential for completion of prophase I during meiotic cell division in male and female germ cells, an unforeseen role for this cell cycle kinase.