Cloning of PCP1, a member of a family of pollen coat protein (PCP) genes from Brassica oleracea encoding novel cysteine-rich proteins involved in pollen-stigma interactions

The pollen coatings of both Brassica oleracea and Brassica napus contain a small family of basic 6–8 kDa proteins which are released on to the stigmatic surface on pollination. Following partial amino-acid sequencing of one of these pollen coat proteins (PCPs), PCR primers were constructed to isolate the PCP sequence from anther mRNA using RT-PCR. A cDNA was obtained which, in Northern hybridization experiments, revealed a characteristic pattern of expression during late stages of anther development. Interestingly, in situ hybridization revealed expression of this sequence to be confined to the cytoplasm of the trinucleate pollen grains: no signal was detected in the tapetum. Southern hybridization experiments have shown the gene ( PCP1 ) to be a member of a large family of between 30 and 40 PCP genes in the genome of Brassica oleracea , Surprisingly, RFLP experiments showed reduced copy number (one to two copies) in some of the F₂ segregants, perhaps resulting from the clustering of PCP sequences. PCP1 contains a single intron and encodes a small, basic peptide 83 amino acids in length featuring a hydrophobic signal peptide sequence separated from the more hydrophilic, cysteine-rich mature protein. The central part and C-terminal region of the peptide contain a characteristic and invariant pattern of eight cysteines which show clear homology with a number of other anther-specific genes; the remainder of the sequence shows little similarity to other sequences on the data bases. The product of PCP1 is a member of a large family of similar proteins, some of which have been demonstrated to bind specifically to S-locus glycoproteins, but does not appear to be genetically linked to the S-locus .     

FLP-mediated site-specific recombination in microinjected murine zygotes

The FLP recombinase of yeast catalyses site-specific recombination between repeated FLP recombinase target (FRT) elements in yeast and in heterologous system (Escherichia coli, Drosophila, mosquito and cultured mammalian cells). In this report, it is shown that transient FLP recombinase expression can recombine and activate an extrachromosomal silent reporter gene following coinjection into fertilized one-cell mouse eggs. Furthermore, it is demonstrated that introduction of a FLP-recombinase expression vector into transgenic one-cell fertilized mouse eggs induces a recombination event at a chromosomal FRT target locus. The resulting event occured at the one-cell stage and deleted a chromosomal tandem array of a FRT containinglacZ expression cassette down to one or two copies. These results demonstrate that the FLP recombinase can be utilized to manipulate the genome of transgenic animals and suggest that FLP recombinase-mediated plasmid-to-chromosome targeting is feasible in microinjected eggs.     

PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells,a new and more cost-effective approach

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where theneo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects.     

Cellular mechanisms governing synapse formation: lessons from identified neurons in culture

The accessibility of embryonic and adult neurons within invertebrate nervous systems has made them excellent subjects for neurobiological study. The ability to readily identify individual neurons, together with their great capacity for regeneration, has been especially beneficial to investigations of synapse formation and the specificity of neuronal connectivity. Many invertebrate neurons survive for long periods following isolation into primary cell culture. In addition, they readily extend new neuritic arbors and form electrical and chemical connections at sites of contact. Thus, cell culture approaches have allowed neuroscientists greater access to, and resolution of, events underlying neurite outgrowth and synaptogenesis. Studies of identified neuromuscular synapses ofHelisoma have determined a number of signaling mechanisms involved in transsynaptic communication at sites of neuron-target contact. At these sites, both anterograde and retrograde signals regulate the transformation of growth cones into functional presynaptic terminals. We have found that specific muscle targets induce both global and local changes in neurotransmitter secretion and intracellular calcium handling. Here we review recent studies of culturedHelisoma synapses and discuss the mechanisms thought to govern chemical synapse formation in these identified neurons and those of other invertebrate species.     

Relationships among didelphid marsupials based on sequence variation in the mitochondrial cytochrome B gene

Variation in the mitochondrial cytochrome b gene (nucleotide and amino acid sequences) is evaluated for 9 genera and 15 species of American opossums in the family Didelphidae, using the American caenolestid rat opossumLestoros and the New Guinean peroryctid bandicootEchimypera as outgroups. Phylogenetic analyses (parsimony and distance) strongly support the monophyly of the Didelphidae and delineate two major clades; (1)Didelphis andPhilander are strongly aligned sister taxa, withMetachirus weakly but consistently associated with them, and (2)Marmosa plusMicoureus, withMonodelphis falling outside that pair. The generaMarmosops, Caluromys, andGlironia exhibit varied relationships, depending upon the method of analysis and data (DNA or amino acid sequences) used, but generally are placed individually or in combinations near or at the base of the didelphid radiation. Some aspects of these relationships are consistent with current taxonomic views, but others are in marked contrast. Specifically, a clade comprised of the mouse opossumsMarmosa, Micoureus, andMarmosops is strongly rejected by log-likelihood analysis, contrary to expectations from some current classifications. Also, the woolly opossumsCaluromys andGlironia also do not form a sister-taxon relationship, as suggested by their placement in a subfamily separate from the remaining didelphids examined. However, such a relationship cannot be rejected from log-likelihood analyses. The relationships suggested fromcyt-b sequences are strongly concordant with those based on DNA-DNA hybridization analyses. In addition to systematic and phylogenetic properties, molecular evolution of the didelphid cytochrome b gene sequence is characterized according to nucleotide bias and rate differentials at each codon position and across the entire sequence.To whom correspondence should be addressed.     

Angiogenic potential of microvessel fragments established in three-dimensional collagen gels

Summary During angiogenesis, the microvasculature displays both vessel remodeling and expansion under the control of both cellular and extracellular influences. We have evaluated the role of angiogenic and angiostatic molecules on angiogenesis in anin vitro model that more appropriately duplicates the cellular and extracellular components of this process. Freshly isolated microvessel fragments from rat adipose tissue (RFMF) were cultured within three-dimensional collagen I gels. These fragments were characterized at the time of isolation and were composed of vessel segments observed in the microvasculature of fatin situ (i.e., arterioles, venules, and capillaries). Fragments also exhibited characteristic ablumenally associated cells including smooth muscle cells and pericytes. Finally, fragments were encased in an extracellular matrix composed of collagen type IV and collagen type I/III. The elongation of microvascular elements was subsequently evaluated using morphologic and immunocytochemical techniques. The proliferation, migration, and elongation of cellular elements in microvessel fragments from rat adipose tissue was dependent on initial fragment density, matrix density, and required serum. Inclusion of endothelial cell growth factors to microvessel fragments from rat adipose tissue 3-D cultures resulted in the accelerated elongation of tube structures and the expression of von Willebrand factor in cells constituting these tubes. Molecules with reported angiostatic capacity (e.g., transforming growth factor and hydrocortisone) inhibited vessel tube elongation. In vitro methods have been developed to evaluate numerous mechanisms associated with angiogenesis, including endothelial cell proliferation, migration, and phenotypic modulation. Microvascular endothelial cell fragments described in this study represent anin vitro population of cells that accurately duplicate thein vivo microcirculatory elements of fat. The proliferation of cells and elongation of microvascular elements subsequently observed in three-dimensional cultures provides anin vitro model of angiogenesis. Microvascular formation in this system results from pre-existing microvessel fragments unlike tube formation observed when cultured endothelial cells are placed in three-dimensional gels. This form of tube formation from cultured endothelium is more characteristic of vasculogenesis. Thus, the formation of microvascular elements from microvessel fragments provides the opportunity to examine the mechanisms regulating angiogenesis in anin vitro system amenable to precise experimental manipulation.     

Reexamination of the morphological evidence for the cohort Epitheria (Mammalia,Eutheria)

Novacek and co-workers recognized a monophyletic clade Epitheria, comprising all eutherians except edentates and the extinct palaeoryctoids, on the basis of two synapomorphies: a stirrupshaped stapes and a foramen ovale enclosed within the alisphenoid. To evaluate this phylogenetic hypothesis, we reexamined the distributions of stapedial morphologies and positions of the foramen ovale across Recent and extinct mammals and nonmammalian cynodonts. The states and distributions of the stapes and forament ovale characters used by Novacek and coworkers were modified by recognizing two stapedial characters (one relating to shape of the crura, the other to the nature of the foramen) and a single, multistate foramen ovale character (within, behind, and lateral to the alisphenoid). The taxon-character matrix used by Novacek (1989, 1992b), substituting our amended stapedial and foramen ovale characters and adding several previously unscored extinct taxa and three new characters, was subjected to a series of PAUP manipulations. Identified among the most parsimonious trees were three major topologies for the base of Eutheria: (1) a polytomy including an Edentata/Ungulata clade, (2) a polytomy with Edentata and Ungulata as separate clades, and (3) Edentata and (when included) Palaeoryctoidea as the successive outgroups to a monophyletic Epitheria. We conclude that topology 2 best reflects the current state of knowledge. An edentate/ungulate clade is supported by three characters (from the mastoid region and subarcuate fossa); however, other morphological studies require modification of the distributions of these characters in xenarthrans and bassal ungulates, thereby eliminating support for this clade. In nearly all manipulations, obtaining a monophyletic Epitheria required that one or two steps be added to the most parsimonious trees. When a monophyletic Epitheria was obtained, it was supported by a triangular stapes and, in some trees, the reappearance of a stapedial artery (lost earlier at the level of Recent therians) and a transpromontorial internal carotid artery. In the most parsimonious trees, a foramen ovale within the alisphenoid was an equivocal synapomorphy of Recent therians or cutherians, and a stapes with strongly convex crura (our state closest to the stirrup-shaped state of Novacek and co-workers) appeared independently within various eutherian lineages. The reduction or loss of the stapedial foramen was identified as an independent event in monotremes and within marsupials and various eutherian lineages.To whom correspondence should be addressed.     

Two-Dimensional Crystallization of Brush Border Myosin I

Brush border myosin-I (BBMI) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells, where it links the core bundle of actin filaments to the plasma membrane. An association of BBMI with anionic phospholipids has been shown to be mediated by a carboxy-terminal domain which is rich in basic amino acids. We have exploited this natural affinity of BBMI for negatively charged lipids to form two-dimensional (2D) crystals of this protein which are suitable for structural analysis by electron crystallographic techniques. The 2D crystals which we have obtained belong to one of two space groups, p22₁2₁or p2. We present here projection maps calculated from images of negatively stained crystals for each of these crystal types to a resolution of 20 Å and show that the asymmetric unit is the same in both crystal types.