New P-element vectors for germ-line transformation of Drosophila melanogaster

1987 ◽  
Vol 3 ◽  
pp. 176
1987 ◽  
Vol 7 (3) ◽  
pp. 973-981
Author(s):  
E P Hoffman ◽  
S L Gerring ◽  
V G Corces

The effect of various types of DNA sequence alterations on the activity of the ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene was studied by P element-mediated germ line transformation. Regions of DNA required for proper expression of the gene under these different conditions were identified. Wild-type levels of transcription during oogenesis are dependent on two elements respectively located within a 64-base-pair (bp) fragment in the transcribed untranslated region and between -227 and -958 bp upstream of the transcription start site. This ovarian expression is particularly sensitive to both chromosomal position effects and an increased distance between the distal upstream promoter element and the TATAA homology. The ecdysterone-mediated expression during metamorphosis is dependent on a 145-bp domain including the TATAA box and additional upstream sequences that augment transcription by two- to five-fold. Finally, sequences necessary for heat shock expression are located much further upstream from hsp27 than those previously found for hsp70, although basal expression was correlated with the presence of more proximal heat shock consensus sequences.


Gene ◽  
1992 ◽  
Vol 114 (2) ◽  
pp. 187-193 ◽  
Author(s):  
Trevor J. Lockett ◽  
Denise Lewy ◽  
Patricia Holmes ◽  
Kerrie Medveczky ◽  
Robert Saint

Genome ◽  
1994 ◽  
Vol 37 (3) ◽  
pp. 356-366 ◽  
Author(s):  
A. Alex Szekely ◽  
R. C. Woodruff ◽  
R. Mahendran

Questions relating to the origin and regulation of mobile genetic elements are currently of considerable interest. Since it is now possible to address more precisely issues concerning the entry, dispersion, and regulation of elements within a virgin genome, one approach that may afford a better understanding of transposable elements in general could be provided by interspecific DNA transformation. Therefore, the Tc1 transposable DNA element from Caenorhabditis elegans was chosen as a proposed invading element of the Drosophila melanogaster genome. The basis for this selection resided in the inherent structural and functional similarities, as well as sequence identities, between the Caenorhabditis element and elements innate to Drosophila (e.g., P, HB1, and Uhu). Initial investigations were carried out to define a clone carrying an intact Tc1 element. This Tc1 element was inserted into a P transposon vector and two P–Tc1–ry+ constructs, differing only in insert orientation, were identified. P element mediated germ line transfer was then used to generate a transformant that was genetically and molecularly identified as containing a single, structurally intact Tc1 element at cytological location 64C4-5 on the third chromosome. The single P[(Tc1, ry+)]SAS-B insertion was thereafter mobilized by using a P[ry+Δ2-3] element as a transposase source, and the genetic and molecular data suggested that the insertion had been successfully reintegrated to a variety of genomic locations. On the basis of genetic and molecular analyses, the Tc1 element in the P[(Tc1, ry+)] transformed stock is not highly unstable in germ line and somatic tissues.Key words: Tc1 transposable element, transformation, Drosophila melanogaster, Caenorhabditis elegans, P elements.


1987 ◽  
Vol 7 (3) ◽  
pp. 973-981 ◽  
Author(s):  
E P Hoffman ◽  
S L Gerring ◽  
V G Corces

The effect of various types of DNA sequence alterations on the activity of the ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene was studied by P element-mediated germ line transformation. Regions of DNA required for proper expression of the gene under these different conditions were identified. Wild-type levels of transcription during oogenesis are dependent on two elements respectively located within a 64-base-pair (bp) fragment in the transcribed untranslated region and between -227 and -958 bp upstream of the transcription start site. This ovarian expression is particularly sensitive to both chromosomal position effects and an increased distance between the distal upstream promoter element and the TATAA homology. The ecdysterone-mediated expression during metamorphosis is dependent on a 145-bp domain including the TATAA box and additional upstream sequences that augment transcription by two- to five-fold. Finally, sequences necessary for heat shock expression are located much further upstream from hsp27 than those previously found for hsp70, although basal expression was correlated with the presence of more proximal heat shock consensus sequences.


Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 749-760 ◽  
Author(s):  
Armin Schmidt ◽  
Gioacchino Palumbo ◽  
Maria P Bozzetti ◽  
Patrizia Tritto ◽  
Sergio Pimpinelli ◽  
...  

Abstract The sting mutation, caused by a P element inserted into polytene region 32D, was isolated by a screen for male sterile insertions in Drosophila melanogaster. This sterility is correlated with the presence of crystals in spermatocytes and spermatids that are structurally indistinguishable from those produced in males carrying a deficiency of the Y-linked crystal (cry) locus. In addition, their morphology is needle-like in Ste+ flies and star-shaped in Ste flies, once again as observed in cry– males. The sti mutation leads to meiotic drive of the sex chromosomes, and the strength of the phenomenon is correlated with the copy number of the repetitive Ste locus. The same correlation is also true for the penetrance of the male sterile mutation. A presumptive sti null allele results in male sterility and lethal maternal effect. The gene was cloned and shown to code for a putative protein that is 866 amino acids long. A C-terminal domain of 82 amino acids is identified that is well conserved in proteins from different organisms. The gene is expressed only in the germline of both sexes. The interaction of sting with the Ste locus can also be demonstrated at the molecular level. While an unprocessed 8-kb Ste primary transcript is expressed in wild-type males, in X/Y homozygous sti males, as in X/Y cry– males, a 0.7-kb mRNA is produced.


1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.


1991 ◽  
Vol 11 (5) ◽  
pp. 2913-2917
Author(s):  
F Maschat ◽  
M L Dubertret ◽  
J A Lepesant

The transcription of the P1 gene is induced by 20-hydroxyecdysone in fat bodies of third-instar larvae. Germ line transformation showed that sequences between -138 to +276 contain elements required for a qualitatively correct developmental and hormonal regulation of P1 transcription. Sequences from -138 to -68 are essential for this expression.


1986 ◽  
Vol 6 (2) ◽  
pp. 663-673 ◽  
Author(s):  
E Hoffman ◽  
V Corces

The transcriptional regulation of the Drosophila melanogaster hsp27 (also called hsp28) gene was studied by introducing altered genes into the germ line by P element-mediated transformation. DNA sequences upstream of the gene were defined with respect to their effect on steroid hormone-induced and heat-induced transcription. These two types of control were found to be separable; the sequences responsible for 80% of heat-induced expression were located more than 1.1 kilobases upstream of the RNA initiation site, while the sequences responsible for the majority of ecdysterone induction were positioned downstream of the site at -227 base pairs. We have determined the DNA sequence of the intergenic region separating hsp23 and hsp27 and have located putative heat shock and ecdysterone consensus sequences. Our results indicate that the heat shock promoter of the hsp27 gene is organized quite differently from that of hsp70.


1991 ◽  
Vol 11 (5) ◽  
pp. 2913-2917 ◽  
Author(s):  
F Maschat ◽  
M L Dubertret ◽  
J A Lepesant

The transcription of the P1 gene is induced by 20-hydroxyecdysone in fat bodies of third-instar larvae. Germ line transformation showed that sequences between -138 to +276 contain elements required for a qualitatively correct developmental and hormonal regulation of P1 transcription. Sequences from -138 to -68 are essential for this expression.


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