Exploiting the complete yeast genome sequence

The completion of the genome sequence of the budding yeast Saccharomyces cerevisiae marks the dawn of an exciting new era in eukaryotic biology that will bring with it a new understanding of yeast, other model organisms, and human beings. This body of sequence data benefits yeast researchers by obviating the need for piecemeal sequencing of genes, and allows researchers working with other organisms to tap into experimental advantages inherent in the yeast system and learn from functionally characterized yeast gene products which are their proteins of interest. In addition, the yeast post-genome sequence era is serving as a testing ground for powerful new technologies, and proven experimental approaches are being applied for the first time in a comprehensive fashion on a complete eukaryotic gene repertoire.     

Identification of the Serratia endonuclease dimer: structural basis and implications for catalysis.

The Serratia endonuclease is an extracellularly secreted enzyme capable of cleaving both single- and double-stranded forms of DNA and RNA. It is the first member of a large class of related and usually dimeric endonucleases for which a structure is known. Using X-ray crystallography, the structure of monomer of this enzyme was reported by us previously (Miller MD et al., 1994, Nature Struct Biol 1:461-468). We now confirm the dimeric nature of this enzyme through light-scattering experiments and identify the physiologic dimer interface through crystal packing analysis. This dimerization occurs through an isologous twofold interaction localized to the carboxy-terminal subdomain of the enzyme. The dimer is a prolate ellipsoid with dimensions 30 A x 35 A x 90 A. The dimer interface is flat and contains four salt links, several hydrogen bonds, and nonpolar interactions. Buried water is prominent in this interface and it includes an unusual "cubic" water cluster. The position of the two active sites in the dimer suggests that they can act independently in their cleavage of DNA, but have a geometrical advantage in attacking substrate relative to the monomer.     

Classification and expression of a family of cyclin gene homologues in Brassica napus

In order to investigate the role of cell division in plant development, we isolated several plant genes which encode homologues of animal and yeast cell cycle regulators known as cyclins.Through the use of degenerate primers and the polymerase chain reaction (PCR) we isolated a Brassica sequence which showed homology to the cyclin box functional domain found within cyclin proteins. Southern blot analysis indicated that Brassica napus has a large number of genes containing cyclin box-related sequences. This was further supported by the isolation of cyclin box sequences from six different genomic clones. In addition, we have isolated two different cyclin cDNA clones, BnCYC1 and BnCYC2, from a Brassica napus shoot apical cDNA library. Both of the cDNA clones contain a destruction box regulatory domain similar to animal mitotic cyclins.Northern blot analysis using BnCYC2 shows mRNA levels which correlate well with the level of cell division in various tissues. Messenger RNA abundance was highest in 1–3 mm leaves, root tips and shoot apices. The mRNA detected using BnCYC1 was restricted to young leaves and the shoot apex, suggesting divergent, organ-specific roles for cyclin family members. The results demonstrate that the plant cyclin gene family is more extensive than previously demonstrated and consists of genes expressed in all dividing tissues as well as a subset of developmentally specific members.     

Effects of manipulation of food supply on estuarine meiobenthos

A comparative mesocosm experiment was carried out to determine the effects of natural foods of different quality and quantity on the structure of natural meiobenthic communities collected in undisturbed sediment from the polluted Westerschelde and the comparatively undisturbed Gironde estuaries. Nematode communities are more diverse and species rich in the latter estuary. The organic matter or foods used were phytoplankton, green alga, salt marsh plant detritus and leaf litter detritus which were added at three dose rates including a high dose. There was no change in community structure in response to the treatments in either of the estuarine meiobenthic communities. Analysis of all the results from this experiment indicate that the food quantity manipulations had almost no effect on the deposit feeding meiofauna. It may be that the reserves of organic matter within the sediment were sufficient to satisfy their dietary requirements for the duration of the experiment. The abundance of diatom/epigrowth feeding nematodes which were initially dominant in the Gironde, declined substantially suggesting that they may have been food limited since diatoms were not among the sources of organic matter added to the mesocosm. There was no specific response to the five different types of organic matter added to the mesocosm     

Membrane potential and input resistance are ambiguous measures of sealing of transected cable-like structures.

For many years, membrane potential (Vm) and input resistance have been used to characterize the electrophysiological nature of a seal (barrier) that forms at the cut end of a transected axon or other extended cytoplasmic structure. Data from a mathematical and an analog model of a transected axon and other theoretical considerations show that steady-state values of Vm and input resistance measured from any cable-like structure provide a very equivocal assessment of the electrical barrier (seal) at the cut end. Extracellular assessments of injury currents almost certainly provide a better electrophysiological measure of the status of plasma membrane sealing because measurements of these currents do not depend on the cable properties of extended cytoplasmic processes after transection.     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Isolation of genomic and cDNA clones encoding bovine poly(A) binding protein II.

cDNA clones for bovine poly(A) binding protein II (PAB II) were isolated. Their sequence predicts a protein of 32.8 kDa, revising earlier estimates of molecular mass. The protein contains one putative RNA-binding domain of the RNP type, an acidic N-terminal and a basic C-terminal domain. Analyses of authentic PAB II were in good agreement with all predictions from the cDNA sequence except that a number of arginine residues appeared to be post-translationally modified. Poly(A) binding protein II expressed in Escherichia coli was active in poly(A) binding and reconstitution of processive polyadenylation, including poly(A) tail length control. The cDNA clones showed a number of potential PAB II binding sites in the 3' untranslated sequence. Bovine poly(A)+RNA contained two mRNAs hybridizing to a PAB II-specific probe. Analysis of a genomic clone revealed six introns in the coding sequence. The revised molecular mass led to a demonstration of PAB II oligomer formation and a reinterpretation of earlier data concerning the protein's binding to poly(A).     

Distribution of androgenetic cells in fetal mouse chimeras

To asses the potential of androgenetic cells to participate in post-midgestation fetal development we have made use of an in situ detectable cell lineage marker in the analysis of chimeric mouse fetuses containing an androgenetic cell lineage. Our results show conclusively that androgenetic cells participate in the formation of derivatives of all lineages and in some tissues may contribute the majority of the total cell population. However, the allocation or persistence of androgenetic cells was non-random. High contribution of androgenetic cells was observed in brown adipose tissue, mesenchyme, smooth muscle, perichondrium, peripheral nerves and epithelia of the intestinal tract and the trachea. Thus, androgenetic cells were able to efficiently populate mesodermal, ectodermal and endodermal derivatives. In contrast, there was a clear prejudice against androgenetic cells in the brain.