Zeev Lev

Rop, a drosophila homolog of yeast Sec1 and vertebrate n-Sect/Munc-18 proteins, is a negative regulator of neurotransmitter release in vivo

The mammalian homolog of the yeast Sec1p, n-Sec1/Munc-18 has been demonstrated to bind the presynaptic membrane protein syntaxin, a putative synaptic vesicle docking protein. To determine the role of n-Sec1/Munc-18 in neurotransmitter release in vivo, we have overexpressed the Drosophila homolog, rop, in third instar larvae and measured the electrophysiological consequences at the neuromuscular junction. A 3- to 5-fold induction of the rop protein causes a dramatic decrease in neurotransmitter release, suggesting rop may restrict the ability of vesicles to dock or of docked vesicles to fuse. Consistent with this hypothesis, rop overexpression also reduces the number of spontaneous vesicle fusions by more than 50%, and repetitive stimulation results in significant decreases in evoked responses similar to those observed in rab3a mutant mice. However, rop overexpression does not alter significantly the Ca2+ dependence of neurotransmitter release. We propose that the Drosophila n-Sec1/Munc-18 homolog plays a negative role in neurotransmitter release in vivo, in addition to its previously identified positive function, possibly by modulation of docking of synaptic vesicles or activation of a pre-fusion complex at the active zone.

The Sec1 family: a novel family of proteins involved in synaptic transmission and general secretion.

Abstract: The Sec1 family, a novel family of proteins involved in synaptic transmission and general secretion, is described. To date, 14 members of this family have been identified: four yeast proteins, Sec1, Sly1, Slp1/Vps33, and Vps45/Stt10; three nematode proteins, Unc‐18 and the homologues of Sly1 and Slp1; the Drosophila Rop; and six mammalian proteins, the rat Munc‐18/n‐Sec1/rbSec1A and rbSec1B, the mouse Munc‐18b/muSec1 and Munc‐18c, and the bovine Munc‐18 and mSec1. The mammalian proteins share 44–63% sequence identity with the nematode Unc‐18 and Drosophila Rop proteins and 20–29% with the yeast proteins and their nematode homologues. The Sec1 proteins are mostly hydrophilic and lack a transmembrane domain. Nevertheless, Sec1 proteins are found as membrane‐bound proteins. Some of them are also found as soluble, cytoplasmic proteins. Binding of the rat brain Sec1 to the presynaptic membrane may be due to strong interaction with syntaxin, an integral component of this membrane. The rat brain Sec1 is also bound to Cdk5, a neural cyclin‐dependent kinase. The Sec1 proteins play a positive role in exocytosis. Loss of function mutations in SEC1, SLY1, or SLP1 result in blocking of protein transport between distinct yeast subcellular compartments. Inactivation of unc‐18 and rop results in inhibition of neurotransmitter release and, in the case of rop, inhibition of general secretion as well. In addition, studies of Rop and n‐Sec1 indicate that they also play a negative role in synaptic transmission, mediated by their interaction with syntaxin. A working model addressing the dual regulative role of the Sec1 proteins in secretion is presented.

Mutations in the β-propeller domain of the Drosophila brain tumor (brat) protein induce neoplasm in the larval brain

Inactivation of both alleles of the fruit fly D. melanogaster brain tumor (brat) gene results in the production of a tumor-like neoplasm in the larval brain, and lethality in the larval third instar and pupal stages. We cloned the brat gene from a transposon-tagged allele and identified its gene product. brat encodes for an 1037 amino acid protein with an N-terminal B-box1 zinc finger followed by a B-box2 zinc finger, a coiled-coil domain, and a C-terminal β-propeller domain with six blades. All these motifs are known to mediate protein–protein interactions. Sequence analysis of four brat alleles revealed that all of them are mutated at the β-propeller domain. The clustering of mutations in this domain strongly suggests that it has a crucial role in the normal function of Brat, and defines a novel protein motif involved in tumor suppression activity. The brat gene is expressed in the embryonic central and peripheral nervous systems including the embryonic brain. In third instar larva brat expression was detected in the larval central nervous system including the brain and the ventral ganglion, in two glands – the ring gland and the salivary gland, and in parts of the foregut – the gastric caecae and the proventriculus. A second brat-like gene was found in D. melanogaster, and homologs were identified in the nematode, mouse, rat, and human. Accumulated data suggests that Brat may regulate proliferation and differentiation by secretion/transport-mediated processes.

K-ras mutations in sporadic colorectal tumors in Israel: unusual high frequency of codon 13 mutations and evidence for nonhomogeneous representation of mutation subtypes.

Colorectal cancer is one of the most common malignancies in the western world, including Israel. An important step in progression includes induction of activating mutations in the protooncogene K-ras. This event is very frequent and is detected in about 40% of colorectal carcinomas. Previous studies of a variety of genetic disorders revealed unique gene mutation prevalence in Jewish populations, attributed both to differences in genetic background and to variability in environmental exposure. To determine the incidence and molecular subtypes of K-ras mutations in colorectal cancer in Israel, compared with other countries, DNA was isolated from a random collection of 105 colorectal carcinoma samples, and K-ras mutations were detected by an improved designed RFLP and direct sequencing. K-ras sporadic mutations in colorectal cancer in Israel are relatively frequent, with a higher fraction in codon 13 than reported thus far. Comparison with other countries shows a vast heterogeneity in terms of the relative abundance of the affected K-ras codon and in type and representation of specific mutations. The heterogeneous distribution found may be due to a variable genetic background and different environmental factors involved in the initiation and propagation of sporadic colorectal cancer.

Utilization of K-ras mutations identified in stool DNA for the early detection of colorectal cancer

Colorectal cancer is one of the most common malignancies in the western world. About 60,000 Americans die of colorectal cancer each year. The annual incidence rate in Israel is 40 per 100,000 persons, namely a total of 2,000 new cases each year. An important step in the progression of colorectal cancer includes induction of activating mutations in the proto‐oncogene K‐ras. The mutations in K‐ras appear early during tumorigenesis, at the intermediate adenoma stage, and thus can be used as a biomarker for early detection in about 40% of colonic tumors. A large yet unknown number of mutated cells are shed from the developing tumor during its progression. Indeed, K‐ras mutations were detected in DNA isolated from stool obtained from symptomatic and asymptomatic patients with colorectal cancer, suggesting a novel approach for a noninvasive screening procedure. However, severe difficulties in obtaining reproducible yields of amplifiable DNA from stool, and usage of nonquantitative, time‐consuming procedures, hampered further progress in the utilization of K‐ras mutations for the early detection of colorectal cancer. Apparently a novel protocol is required that provides reproducible output of amplifiable DNA from small amounts of stool, detects if K‐ras mutated DNA is present, and determines the quantity of K‐ras mutated cells in the stool sample. In addition, this protocol should be simple, robotics compatible, and thus suitable for cost‐effective, large‐scale mutation screening. Molecular assays for detecting K‐ras mutations and additional biomarkers in stool DNA promise to be highly sensitive, specific, and cost‐effective. As such they should be very effective when used in chemoprevention studies and screening protocols for colorectal cancer. J. Cell. Biochem. Suppl. 34:35–39, 2000.

Developmental changes in expression of the Drosophila melanogaster epidermal growth factor receptor gene.

A Drosophila gene homologous to the human EGF receptor gene has recently been isolated and sequenced. Two transcripts, 7.6- and 7.1-kb long, encoded by this gene were identified in Drosophila melanogaster. The transcripts are present at low abundance in the maternal RNA stored in unfertilized eggs and in 2-hr-old embryos. The abundance of both transcripts increases sharply between 2 and 5 hr after egg deposition, it remains high throughout embryogenesis, and decreases again in the larval and pupal stages. In adult flies the two transcripts are expressed differentially. The 7.1-kb transcript is present during adulthood at the same level detected previously in pupal stage, but the abundance of the 7.6-kb transcript decreases substantially and it remains low during adulthood.

A procedure for large-scale isolation of RNA-free plasmid and phage DNA without the use of RNase

A preparative procedure for the large-scale isolation of plasmid DNA without the use of RNAse is described. Crude plasmid DNA is prepared using a standard boiling method. High-molecular-weight RNA is removed by precipitation with LiCl, and low-molecular-weight RNA is removed by sedimentation through high-salt solution. The procedure is inexpensive, rapid, simple, and particularly suitable for processing several large-scale preparations simultaneously. A similar procedure has been developed for preparation of lambda-phage DNA.

The RNA transcripts of Drosophila melanogaster src gene are differentially regulated during development

Abstract This article describes the expression of the Drosophila src gene located at 64B. The gene extends over at least 7 kb of genomic DNA, and codes for three transcripts which are 3.0, 4.4 and 4.8 kb long. Their 3′ ends were identified and tentatively located on the physical map of the gene. All three transcripts are in the same polarity and contain the information required for translating the protein kinase domain common to all members of the src gene family. The Drosophila src transcripts are expressed in two different patterns during development. The 3.0 and 4.4 transcripts are abundant in unfertilized eggs and during embryogenesis. Their prevalence decreases under the limit of detection in the larval and pupal stages, and they reappear in adult flies. The larger 4.8 kb transcript is undetectable in unfertilized eggs. It starts accumulating between 2 and 5 h after fertilization, and is expressed continuously during development.

Structure of the Drosophila ras2 Bidirectional Promoter

In D.melanogaster three ras genes were isolated and mapped to regions 85D, 64B and 62B, respectively (Neuman-Silberberg et al, 1984). Nucleotide sequence analysis of genomic and cDNA clones suggests that the putative Drosophila ras proteins have a molecular weight of 21 to 23 kd. They share on average 80 percent similarity with vertebrate ras p21 proteins at their N-terminus and 40 percent at their C-terminus. The specific pattern of sequence conservation is similar to that found when vertebrate ras genes are compared among themselves or with yeast ras genes, probabely reflecting different domains within the ras protein (Neuman-Silberberg et al, 1984; Mozer et al, 1985; Brock, 1987). Since the Drosophila rani. gene is more similar to vertebrate ras genes than to the Drosophila ras2 gene, it has been proposed that the two Drosophila genes have diverged anciently and that they have different functions (Brock, 1987). The structural and functional homologies between the Drosophila and vertebrate ras proteins were demonstrated by precipitating 21 and 27 to 28 kd proteins from Drosophila cell extract using monoclonal antibodies raised against the vHa-ras p21 protein (Papageorge et al, 1984), and by transforming rat cells with a chimeric ras comprising the N-terminus of human activated Ha-ras gene and the C-terminus of Drosophila ras gene (Schejter et al, 1985).

Topography of polyoma virus-specific giant nuclear RNA molecules containing poly(A) sequences.

Abstract The distribution of viral RNA sequences with respect to poly(A) tracts has been determined for giant polyoma virus-specific nuclear RNA molecules. Polyadenylated nuclear RNA sedimenting faster than 30 S was isolated from polyoma virus-infected cells and cleaved into fragments of various sizes by partial alkaline hydrolysis. Fragments containing poly(A) were separated from nonpolyadenylated RNA. The viral sequence composition of polyadenylated or nonpolyadenylated fragments, and of total polyadenylated giant RNA, was determined by hybridization to 32P-labeled separated strands of specific restriction endonuclease fragments of polyoma virus DNA. The results of this analysis showed that in all polyadenylated giant RNA molecules transcribed from the L DNA strand the portion adjacent to the poly(A) segment hybridizes to the 3′-terminal half of the late region of the viral genome. The next portion of these chains hybridizes to the remainder of the late region, the more distal part hybridizes to the early region, and the portions furthest from the poly(A) contain sequences complementary to the L DNA strand of both early and late regions. This finding shows that polyadenylation of Py giant nuclear RNA is non-random and further suggests that the poly(A)-linked RNA sequence(s) maps within the same region of the viral genome as the poly(A)-linked sequence found in mature cytoplasmic mRNA.