Renate Faast

Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A/E and E2F activities

Pluripotent cells of embryonic origin proliferate at unusually rapid rates and have a characteristic cell cycle structure with truncated gap phases. To define the molecular basis for this we have characterized the cell cycle control of murine embryonic stem cells and early primitive ectoderm-like cells. These cells display precocious Cdk2, cyclin A and cyclin E kinase activities that are conspicuously cell cycle independent. Suppression of Cdk2 activity significantly decreased cycling times of pluripotent cells, indicating it to be rate-limiting for rapid cell division, although this had no impact on cell cycle structure and the establishment of extended gap phases. Cdc2-cyclin B was the only Cdk activity that was identified to be cell cycle regulated in pluripotent cells. Cell cycle regulation of cyclin B levels and Y15 regulation of Cdc2 contribute to the temporal changes in Cdc2-cyclin B activity. E2F target genes are constitutively active throughout the cell cycle, reflecting the low activity of pocket proteins such as p107 and pRb and constitutive activity of pRb-kinases. These results show that rapid cell division cycles in primitive cells of embryonic origin are driven by extreme levels of Cdk activity that lack normal cell cycle periodicity.

Histone variant H2A.Z is required for early mammalian development

Fundamental to the process of mammalian development is the timed and coordinated regulation of gene expression. This requires transcription of a precise subset of the total complement of genes. It is clear that chromatin architecture plays a fundamental role in this process by either facilitating or restricting transcription factor binding [1]. How such specialized chromatin structures are established to regulate gene expression is poorly understood. All eukaryotic organisms contain specialized histone variants with distinctly different amino acid sequences that are even more conserved than the major core histones [2]. On the basis of their highly conserved sequence, histone variants have been assumed critical for the function of mammalian chromatin; however, a requirement for a histone variant has not been shown in mammalian cells. Mice with a deletion of H1 degrees have been generated by gene targeting in ES cells, but these mice show no phenotypic consequences, perhaps due to redundancy of function [3]. Here we show for the first time that a mammalian histone variant, H2A.Z, plays a critical role in early development, and we conclude that this histone variant plays a pivotal role in establishing the chromatin structures required for the complex patterns of gene expression essential for normal mammalian development.

Cdk6-cyclin D3 activity in murine ES cells is resistant to inhibition by p16(INK4a).

Through a screen aimed at identifying genes that are specifically upregulated in embryomic stem (ES) cells but not primitive ectoderm, we identified cyclin D3. This was surprising since cyclin D activity is generally believed to be inactive in ES cells even though retinoblastoma tumor suppressor protein (pRb) accumulates in a predominantly hyperphosphorylated state. Cdk6 is the major catalytic partner for cyclin D3 in ES cells and exhibits robust pRb kinase activity that is downregulated during the early stages of ES embryoid body differentiation. To investigate the basis underlying the insensitivity of ES cells to ectopic p16 expression, we show that Cdk6–cyclin D3 complexes are not subject to inhibition by p16, similar to Cdk–viral cyclin complexes. These observations show that specificity exists between Cdk4/6–cyclin D complexes and their ability to be targeted by p16. Our data suggest that Cdk6–cyclin D3 activity in other cell types, including tumors, may also be refractory to p16-mediated growth inhibition and raises the possibility of additional specificity within the INK4 family.

Implications of pollination by food and sexual deception for pollinator specificity, fruit set, population genetics and conservation of Caladenia (Orchidaceae)

Caladenia is very unusual in that it contains species that attract pollinators by two different strategies, food and sexual deception. Among the sexually deceptive species, baiting for pollinators has shown that within populations orchid species are typically pollinated by a single species of thynnine wasp. However, some wasp species can be pollinators of more than one species of orchid usually when their ranges do not overlap. There is a trend for closely related orchids to exploit wasps from the same genus, with different lineages of orchids often pollinated by different genera. Very little is known about pollination of food-deceptive Caladenia species, although it is evident they attract a suite of generalist food-seeking insects. Food-deceptive species have a higher pollination rate than do sexually deceptive species. Studies of population genetics and pollen movements are few, although they suggest a pattern of fine-scale genetic structuring within populations, owing to predominantly restricted seed dispersal and low genetic differentiation among populations as a consequence of rare long-distance seed-dispersal events. Both evolutionary and ecological research of Caladenia will greatly benefit from a better understanding of the insect species involved in pollination, their ecological requirements and the ecological and genetic consequences of food and sexual deception.

Nucleotide sequence of the structural gene, tcpA, for a major pilin subunit of Vibrio choleras

The toxin co-regulated pilus (Tcp) of Vibrio cholerae appears to be a major protective antigen. By cosmid cloning we have isolated a number of clones capable of converting Tcp- El Tor strains of V. cholerae to Tcp+. A synthetic oligodeoxyribonucleotide probe based upon the N-terminal amino acid sequence of TcpA, has been used to localize the structural gene within the cosmid clones. Using suitable subclones, the nucleotide sequence of the tcpA gene has been determined. The gene encodes a 23.3-kDa pre-protein which in its mature form has a size of 20.3 kDa. The N-terminal leader peptide or signal sequence is atypical and does not conform with the usual rules of such sequences. The TcpA protein shows some similarities to the major pilins of the methylated phenylalanine type or type-4 pili from other bacteria; however, it is sufficiently different that it may represent a new class.

Efficient Generation of α(1,3) Galactosyltransferase Knockout Porcine Fetal Fibroblasts for Nuclear Transfer

Pigs are currently considered the most likely source of organs for human xenotransplantation because of anatomical and physiological similarities to humans, and the relative ease with which they can be bred in large numbers. A severe form of rejection known as hyperacute rejection has been the major barrier to the use of xenografts. Generating transgenic pigs for organ transplantation is likely to involve precise genetic manipulation to ablate the α(1,3) galactosyltransferase (galT) gene. In contrast to the mouse, homologous recombination in livestock species to ablate genes is hampered by the inability to isolate functional embryonic stem cells. However, nuclear transfer using genetically targeted cultured somatic cells provides an alternative means to producing pigs deficient for galT. In this study we successfully produced galT+/− somatic porcine fetal fibroblasts using two approaches; positive negative selection (PNS) using an isogenic targeting construct, and with a promoterless vector using non-isogenic DNA.

The Lectin Dolichos Biflorus Agglutinin Recognizes Glycan Epitopes on the Surface of Murine Embryonic Stem Cells: A New Tool for Characterizing Pluripotent Cells and Early Differentiation

Cell surface markers are key tools that are frequently used to characterize and separate mixed cell populations. Existing cell surface markers used to define murine embryonic stem cells (mESCs) such as stage‐specific embryonic antigen 1 (SSEA1), Forssman antigen (FA), alkaline phosphatase (AP), and CD9 are limiting, however, because they do not unambiguously define the pluripotent state and are not reliable indicators of differentiation commitment. To identify glycan cell surface markers that would circumvent this problem, we used a panel of 18 lectins to identify epitopes specifically elevated on the surface of mESCs, which, during differentiation, decrease with kinetics that precede currently used markers such as CD9, SSEA1, FA, and AP. The anticipated outcome of this analysis was to identify glycans that have utility as reliable mESC markers and high‐resolution readouts for early differentiation commitment. Here, we show that the lectin Dolichos biflorus agglutinin (DBA) recognizes α‐N‐acetylgalactosamine (GalNAc) cell surface epitopes on mESCs (CD9high SSEA1high APhigh DBAhigh). These glycan epitopes decline markedly in cells undergoing the first definable step of differentiation, the transition from mESCs to primitive ectoderm (CD9high SSEA1high APhigh DBAlow). Loss of GalNAc epitopes is, therefore, the earliest cell surface change that can be assigned to differentiating cells, and the only cell surface marker known to be tightly associated with the pluripotent state. The lectin DBA is, therefore, a useful tool to characterize mESC cultures by nondestructive approaches, an indicator of differentiation commitment, and a predictor of developmental potency.

Comparison of the promoter proximal regions of the toxin-co-regulated tcp gene cluster in classical and E1 Tor strains of Vibrio cholerae O1

A physical map has been constructed of the 5-kb XbaI fragment encoding the promoter proximal of region the tcp gene cluster encoding the toxin-coregulated pilus (TCP) of Vibrio cholerae. This fragment contains the major regulatory regions for TCP. Comparison of the nucleotide (nt) sequences from strains of the classical and El Tor biotypes demonstrates that the regions are essentially identical, with several notable exceptions. The intergenic regions, between tcpI and tcpP, and between tcpH and tcpA, show significant sequence divergence which may account for the biotype-related differences in TCP, since this is the location of the major promoter sequences. The C-terminal coding regions of the major pilin subunit, TcpA, also differ. Southern hybridization analyses suggest that the tcpA nt sequence is conserved within a biotype, and Western blot analysis suggests that the two forms of TcpA are antigenically different, but related. Besides tcpA, tcpB, tcpH and tcpI, the genes encoding two additional proteins, TcpP and TcpQ, but not previously defined, were also identified. TcpH and TcpI have been previously suggested to be regulatory proteins but homology data imply that TcpI is a methyl-accepting chemotaxis protein (MCP), as recently reported [Harkey et al., Infect. Immun. 62 (1994) 2669-2678], and TcpH is predicted to be a periplasmic or exported protein. TcpP is thought to be a trans-cytoplasmic membrane (CM) protein which may have a regulatory role.

In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts.

The in vitro development of porcine nuclear transfer embryos constructed using primary cultures from day 25 fetal fibroblasts which were either rapidly dividing (cycling) or had their cell‐cycle synchronized in G0/G1 using serum starvation (serum‐starved) was examined. Oocyte‐karyoplast complexes were fused and activated simultaneously and then cultured in vitro for seven days to assess development. Fusion rates were not different for either cell population. The proportion of reconstructed embryos that cleaved was higher in the cycling group compared to the serum‐starved group (79 vs. 56% respectively; P < 0.05). Development to the 4‐cell stage was not different using either population. Both treatments supported similar rates of development to the morula (1.5 vs. 7%, cycling vs. serum‐starved) and blastocyst stage (1.5 vs. 3%, cycling vs. serum‐starved). The blastocyst produced using cycling cells had a total cell number of 10. Total cell numbers for the three blastocysts produced serum‐starved cells were 22, 24, and 33. These blastocysts had inner cell mass numbers of 0, 15, and 4, respectively. Six hundred and thirty‐five nuclear transfer embryos reconstructed using serum‐starved cells were transferred to 15 temporarily mated recipients for 3–4 days. Of these, 486 were recovered (77% recovery rate) of which 106 (22%) had developed to the 4‐cell stage or later. These were transferred to a total of 15 recipients which were either unmated or mated. Seven recipients farrowed a total of 51 piglets. Microsatellite analysis revealed that none of these were derived from the nuclear transfer embryos transferred. Mol. Reprod. Dev. 57:262–269, 2000.

Investigating DNA barcoding options for the identification of Caladenia (Orchidaceae) species.

The application of molecular techniques for defining evolutionary units in Caladenia has largely focussed on addressing relationships at the subgeneric and deeper levels. However, in light of the morphological complexity present in this diverse genus, molecular markers offer additional characters for the refinement of taxonomy at the species level. In the present study, we explored the utility of marker systems with demonstrated application for defining fine-scale units, both in terms of phylogenetic information and in the context of DNA barcoding. We also provide a working example of the use of molecular techniques for identifying the source plants of pollinia collected from passively sampled pollinators and for identifying sterile plants.