Isolation and characterization of simple sequence repeat loci in the gray tree frog, Hyla chrysoscelis.

A gray tree frog (Hyla chrysoscelis) genomic library was constructed and characterized with regard to the incidence and complexity of simple sequence repeat (SSR) loci. The partial genomic library, containing approximately 10,000 clones with an average-sized insert of 350 bp, was screened with six SSR repeat oligonucleotides (AC, AG, ACG, AGC, AAC, and AAG). Screening identified 31 unique positive clones containing 41 SSR loci. Sequences of tandemly arrayed dinucleotide repeats were more common (36 of 41) than trinucleotide repeats. Twenty-six loci were identified using the AC dinucleotide probe, while 7 loci were identified using the AG dinucleotide probe. An additional 3 AT dinucleotide loci were serendipitously identified. The AT repeats generally comprised the longest dinucleotide repeat loci. The SSR repeat loci reported here should provide potent markers for identity, parentage, and short-lineage determinations in large-scale experiments using gray tree frogs.

Isolation and cloning of Ser4, a gene encoding a trypsin-like serine protease in Drosophila melanogaster.

A gene encoding a unique serine protease in Drosophila melanogaster was characterized using overlapping cDNA and genomic clones. The sequence contains an open reading frame of 798 base pairs encoding a predicted protein 265 amino acids in length with significant homology to other serine proteases. The deduced amino acid sequence of this protein contains several structural features which are highly conserved in active serine proteases, including conserved cysteine and active site residues. Northern blot analysis reveals that the mRNA for the gene is expressed abundantly in the larval gut, suggesting a role in digestion for this protein. Using in situ hybridization to polytene chromosomes, we have localized this gene to region 56E3 on chromosome 2R.

High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

High-molecular-weight proteins often blot onto nitrocellulose membranes poorly following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting in low levels of detection on immunoblots, and hence difficulty in analyzing rare proteins. Moreover, optimizing conditions for the transfer of high-molecular-weight proteins to nitrocellulose frequently results in the inefficient transfer or loss of lower molecular weight proteins. This problem is particularly vexing during the analysis of large proteins which may be processed to one or more smaller biologically active forms. Using radiolabeled protein standards and phosphorimaging technology, we have quantitated the efficacy of many different protein gel electrophoresis and blotting protocols. Here we report novel gel and blotting conditions which significantly improve the transfer and retention of high-molecular-weight proteins, without sacrificing the efficient transfer of lower molecular weight proteins. Using this newly described procedure, we have detected a rare 500-kDa protein in immunoblots which was previously not detectable with any of the commonly used blotting procedures. Since the improved conditions offer increased sensitivity across a spectrum of protein sizes, they should be widely applicable.

Venous stasis: successful outcome, and symptomatic relief in patients undergoing Linton procedures.

A retrospective study of forty-one patients who had undergone subfascial ligation of perforating veins in the lower extremity, or Linton procedure, is presented. All procedures were performed by the same surgeon from 1971 to 1993. Two patients underwent bilateral procedures. There were 28 female patients and 13 males. Fifteen had a history of smoking. There was a history of previous thrombophlebitis in 21 patients, and 21 had a family history of varicosities and/or ulceration. Results of venograms and Doppler duplex studies, if done, were evaluated. Follow up procedures, such as sclerosing injections and skin grafts, were also noted. A total of 25 patients had subsequent sclerosing injections, one underwent debridement twice, and one received skin grafts, secondary to incisional skin necrosis. It was noted that there was no occurrence of wound infection or incisional necrosis when zinc oxide paste boots were applied and maintained post-operatively. All patients were contacted directly prior to the writing of this article to ascertain prevalence, severity, and circumstances of recurrence.

The fat tumor suppressor gene in Drosophila encodes a novel member of the cadherin gene superfamily.

Recessive lethal mutations in the fat locus of Drosophila cause hyperplastic, tumor-like overgrowth of larval imaginal discs, defects in differentiation and morphogenesis, and death during the pupal stage. Clones of mutant cells induced by mitotic recombination demonstrate that the overgrowth phenotype is cell autonomous. Here we show that the fat locus encodes a novel member of the cadherin gene superfamily: an enormous transmembrane protein of over 5000 amino acids with a putative signal sequence, 34 tandem cadherin domains, four EGF-like repeats, a transmembrane domain, and a novel cytoplasmic domain. Two recessive lethal alleles contain alterations in the fat coding sequence, and the dominant fat allele, Gull, contains an insertion of a transposable element in the 33rd cadherin domain. Thus, this novel member of the cadherin gene superfamily functions as a tumor suppressor gene and is required for correct morphogenesis.

Transcripts of the Drosophila blastoderm-specific locus, terminus, are concentrated posteriorly and encode a potential DNA-binding finger.

The commitment of cells to specific fates, as well as the transitions in the cell cycle and transcription that occur at the cellular blastoderm stage of Drosophila embryogenesis, suggest that there are genes with unique functions expressed specifically at this stage. In an attempt to identify such genes, we used molecular screening to isolate several loci which encode blastoderm-specific transcripts (Roark et al., (1985). Dev. Biol. 109, 476-488). We report here the complete nucleotide sequence of one of these genes, terminus (ter), which maps to 75C1,2. The predicted ter protein possesses a transcription factor IIIA (TFIIIA)-like putative Zn-binding, DNA-binding finger. The ter RNA, detected by in situ hybridization, is distributed uniformly in the embryo during the syncytial blastoderm stage, and then becomes more concentrated in the posterior during the late cellular blastoderm stage. During gastrulation, the RNA is most concentrated in the amnioproctodeal invagination; it is also found at a lower concentration in the ventral furrow and in the anterodorsal neurogenic region. By the end of germ band extension, ter RNA is no longer detected.

The zygotic segmentation mutant tailless alters the blastoderm fate map of the Drosophila embryo.

The well-characterized spatial distributions of the transcripts from several Drosophila segmentation genes provide molecular markers which can be used to examine the determination of the segment pattern in early embryos. Tailless (tll) is a zygotic lethal mutation, the phenotype of which is observed by 9 hr of embryogenesis and includes the absence of segments A8, A9, and A10 and a decrease in the procephalic lobe (Strecker et al., Dev. Biol. 113, 64-76, 1986). To establish whether this effect of the tll mutation is due, as proposed previously by Strecker et al., to a reprogramming of the blastoderm fate map, we hybridized probes for the segmentation genes fushi tarazu (ftz) and hairy (h) to whole embryos. The transcripts of these genes show an altered distribution in tll embryos as early as nuclear cycle 14, indicating that the tll gene acts on cellular determination at the blastoderm stage, and is required for normal expression of the ftz and h genes. We obtained more precise information about the alterations in the blastoderm fate map by measuring the position of ftz protein stripes in wild-type and tll embryos. From the results reported here and previously, we conclude that the tll mutation results in a deletion of anterior and posterior ectodermal positional values, concomitant with an expansion of the remaining fate map.

Molecular characterization of bsg25D: a blastoderm-specific locus of Drosophila melanogaster.

The blastoderm stage of Drosophila embryogenesis is a time of crucial transitions in RNA transcription, the cell cycle and segment determination. We have previously identified three loci encoding RNAs specific to this stage (Roark et al., Dev. Biol. 109, 476-488, 1985). We present here the complete nucleotide sequence of one of these loci, bsg25D, which encodes a 2.7 kb blastoderm-specific RNA. The primary structure of this RNA, and that of an overlapping 4.5 kb RNA, has been determined. The amino acid sequence of the predicted bsg25D protein has been compared to the NBRF protein database. Structural similarities between domains in the bsg25D, fos, and tropomyosin proteins, and their possible significance for early embryogenesis are discussed.

Blastoderm-differential and blastoderm-specific genes of Drosophila melanogaster.

We have isolated, by molecular cloning, genes expressed differentially at the blastoderm stage of Drosophila melanogaster. Two of the blastoderm-differential genes are reexpressed at later stages, and map to single chromosomal loci 95C and 99E. The sequence at 99E is that encoding the myosin light chain 2. Two other blastoderm-differential sequences are members of multigene families (one of which is B104, or roo) and map to multiple dispersed chromosomal loci. A gastrula-differential sequence was found which maps to 71A. Most significantly, we have identified three genes encoding transcripts expressed uniquely at the blastoderm stage; these map to single chromosomal loci: 25D3, 75C, and 99D4-8. At least some of the blastoderm-differential and blastoderm-specific loci appear to be distinct from loci involved in embryonic pattern formation that have been identified in recent genetic "saturation" screens. The procedure of identifying genes specific to the blastoderm stage may thus allow the identification of genes, not previously identified by classical genetic techniques, that are involved in important embryonic processes.