A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones.

The recent development of yeast artificial chromosome (YAC) vectors has provided a system for cloning fragments that are over ten times larger than those that can be cloned in more established systems. We have developed a method for the rapid isolation of terminal sequences from YAC clones. The YAC clone is digested with a range of restriction enzymes, a common linker is ligated to the DNA fragments and terminal sequences are amplified using a vector specific primer and a linker specific primer. Sequence data derived from these terminal specific products can be used to design primers for a further round of screening to isolate overlapping clones. The method also provides a convenient method of generating Sequence Tagged Sites for the mapping of complex genomes.     

A 3.5 genome equivalent multi access YAC library: construction, characterisation, screening and storage.

The construction of a yeast artificial chromosome (YAC) primary gridded library of 35,000 clones from human lymphoblastoid (48,XXXX) cell line DNA is described. The average YAC size is approximately 350kb representing a greater than 3.5 times coverage of the genome. The library is stored at -70 degrees C as gridded clones on nylon filters impregnated with 20% glycerol and as glycerol suspensions of individual clones in microtitre plates providing a prolonged multi-user potential. To date we have used 14 single copy probes to screen this library by colony hybridisation as well as PCR and have isolated between 1 and 5 YAC clones for every probe.     

The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog.

WHI1-1 is a dominant mutation that reduces cell volume by allowing cells to commit to division at abnormally small sizes, shortening the G1 phase of the cell cycle. The gene was cloned, and dosage studies indicated that the normal gene activated commitment to division in a dose-dependent manner, and that the mutant gene had a hyperactive but qualitatively similar function. Mild over-expression of the mutant gene eliminated G1 phase, apparently entirely relaxing the normal G1 size control, but revealing hitherto cryptic controls. Sequence analysis showed that the hyperactivity of the mutant was caused by the loss of the C-terminal third of the wild-type protein. This portion of the protein contained PEST regions, which may be signals for protein degradation. The WHI1 protein had sequence similarity to clam cyclin A, to sea urchin cyclin and to Schizosaccharomyces pombe cdc13, a cyclin homolog. Since cyclins are inducers of mitosis, WHI1 may be a direct regulator of commitment to division. A probable accessory function of the WHI1 activator is to assist recovery from alpha factor arrest; WHI1-1 mutant cells could not be permanently arrested by pheromone, consistent with a hyperactivation of division.     

Effect of Within-field Variation in Soil Texture on Heterodera glycines and Soybean Yield

The influence of soil texture on Soybean yield in the presence of Heterodera glycines was investigated by comparing yields of susceptible cultivars with a resistant cultivar for 2 years. Soybean yield was negatively correlated with increasing sand content (P = 0.05). Yields of susceptible cultivars were suppressed with increasing sand content. Final nematode population densities were lowest in plots with greatest sand content. Soybean infection by SCN, as determined by the number of cysts 30 days after planting, was not consistently related to soil texture over 2 years. Initial nematode population density was positively related to soybean yield the first year and negatively related to soybean yield the second, probably a result of greater yield suppression by H. glycines in plots with greater sand content.     

N2(C2H2−C2H4) fixation in two species of Ceanothus seedlings in second year postfire chaparral

Nitrogen fixation in excised root nodules of 2-year-old, postfireCeanothus tomentosus andC. leucodermis seedlings was measured over an 8-month period using the acetylene reduction method. High levels of NO₃–N and NH₄–N present in postfire soils were limited to the upper 10 cm and did not inhibit nodulation in these deeper-rooting seedlings. Decreases in acetylene reduction activity occurred with decreased soil moisture and increased soil temperature. Nitrogen gains from these two Ceanothus shrub seedlings totalled 1.6 kg N ha^–1 yr^–1.     

Computer-designed prostheses for orbitocranial reconstruction

Three-dimensional imaging is an adjunct to preoperative evaluation and surgical management in some patients with complex anatomic defects of various etiologies. Deformities defined by conventional computerized tomography can be viewed as accurate three-dimensional images calculated from the original scan. The images are viewed on a high-resolution video monitor and can be photographed for a permanent record. A computer-controlled milling device can use these data to fabricate prostheses. The prostheses aid reconstructive surgery through use as an alloplastic implant, as a template to fashion autogenous bone grafts, or as a model for tissue removal. We have utilized three-dimensional imaging in combination with computer-assisted prosthesis manufacture in six patients with complex orbitocranial deformities. Four patients have undergone reconstructive surgery with satisfactory results and no complications thus far. The use of computer-designed prostheses adds a new aspect to orbitocranial reconstructive surgery that facilitates increased accuracy in the correction of anatomic defects.     

Truncation of the ectodomain of the human insulin receptor results in secretion of a soluble insulin binding protein from transfected CHO cells

The insulin receptor is an integral transmembrane glycoprotein comprised of two alpha-(approximately 135 kDa) and two beta-(approximately 95 kDa) subunits, which is synthesized as a single polypeptide chain precursor (alpha beta). The primary sequence of the human insulin receptor (hIR) protein, deduced from the nucleotide sequence of cloned human placental mRNAs, predicts two large domains (929 and 403 residues) on either side of a single membrane spanning domain (23 residues); each of these major domains has a distinct function (insulin binding and protein/tyrosine kinase activity, respectively). To experimentally test this deduced topology, and to explore the potential for independent domain function by the hIR extracellular domain, we have constructed an expression plasmid encoding an hIR deletion mutant which is truncated 8 residues from the beginning of the predicted transmembrane domain (i.e., 921 residues). This domain of the hIR is in fact processed into alpha- and truncated beta-subunits and secreted with high efficiency from transfected CHO cell lines which express this mutant hIR, and the protein accumulates as an (alpha beta)2 dimer in the medium. This molecule is recognized by a battery of 13 monoclonal antibodies to epitopes on the IR extracellular domain, four of which block insulin binding and two of which require the native conformation of the IR for recognition. Further, this domain binds insulin with an apparent dissociation constant comparable to that of the wild-type hIR. However, the secreted dimer displays a linear Scatchard plot, while that of the wild-type membrane-associated hIR is curvilinear.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructure of the pronephric kidney of embryos and prolarvae of the sea lamprey, Petromyzon marinus

Embryos of lampreys Petromyzon marinus were obtained through a technique of artificial fertilization. Samples of developmental intervals to the prolarval stage were prepared for transmission electron microscopy and the pronephros was examined. The pronephros was visible in the cardiac region of the coelom prior to the time of hatching of embryos and consisted of a renal corpuscle, nephrostomes, and proximal tubules connected to a pronephric duct. The renal corpuscle was comprised of poorly-defined vascular channels and a visceral epithelium of yolk-filled cells, the podocytes, with short major processes and pedicels resting on a basal lamina. The first proximal tubules possessed a delicate brush border of short microvilli but subsequent cellular differentiation yielded cells with all the components required for the process of endocytosis, a process which was demonstrated by uptake of the tracer, horseradish peroxidase. The distal tubules appeared later in development and were noted for abundant mitochondria and an extensive smooth tubular network. The timing of differentiation of various components of the nephron corresponds to that seen during morphogenesis of other vertebrate kidneys.     

Structure of equine corticotropin releasing factor

A 41 amino acid peptide, probably identical in structure to human corticotropin releasing factor, was isolated from 70 equine hypothalami by methanol extraction, immunoaffinity chromatography and single step of reverse phase HPLC. The amino acid sequence was determined by gas phase sequence analysis. Probable carboxyl terminal amidation was demonstrated by similar retention times for equine and human corticotropin releasing factor on reverse phase HPLC at pH 8. The likely structure of equine corticotropin releasing factor is: Ser-Glu-Glu-Pro-Pro- Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn- Arg-Lys-Leu-Met-Glu-Ile-Ile-NH₂. The purified peptide is equipotent with human corticotropin releasing factor in an in vitro bioassay and in a human plasma binding protein assay.     

Calretinin and calbindin in the retina of the developing chick

Summary Calretinin and calbindin-D28k are two calcium-binding proteins that are present in largely different sets of nerve cells in the central nervous system. Their appearance during development of the chick retina was studied by immunohistochemistry and Western blots. The patterns are mature one day before hatching. Each cell type acquires its characteristic calcium-binding protein several days after its differentiation has started, but in most cases before morphological maturation is complete. There is also an early phase of calbindin immunoreactivity in many immature amacrine cells, and of calretinin immunoreactivity in the presumptive photoreceptor layer, suggesting that these proteins may have distinct functions in differentiating cells.Abbreviations CR+ Immunoreactive for calretinin only - CB+ immunoreactive for calbindin only - CR+CB+ immunoreactive for both antisera - IPL inner plexiform layer - OPL outer plexiform layer