Isolation of three novel Drosophila melanogaster genes containing repetitive sequences rich in GCN triplets

Genome ◽  
1992 ◽  
Vol 35 (1) ◽  
pp. 133-139
Author(s):  
Charalambos Magoulas ◽  
Donal A. Hickey
Keyword(s):  
Drosophila Melanogaster ◽  
Cdna Clone ◽  
Sequence Similarity ◽  
Repetitive Sequences ◽  
Cdna Clones ◽  
Drosophila Genome ◽  
Significant Sequence Similarity ◽  
Reading Frame ◽  
Genomic Clones ◽  
Gene Libraries

Several cDNA and genomic clones were isolated from Drosophila melanogaster gene libraries by hybridization with a region of a mammalian gene that contains a simple repetitive sequence of six GCN repeats. One of the cDNA clones, E6, was completely sequenced and it was shown that it contains a region of 16 GCN repeats; these repeats encode a polyalanine stretch within a long open reading frame. The sequencing of three different genomic clones (A, B, and D) revealed that all the isolated Drosophila clones are similar to one another in a short region containing variable numbers of the GCN repeat. The genomic clone B was found to be the genomic counterpart of the cDNA clone E6. The other genomic clones, A and D, also hybridize with Drosophila cDNA clones at high stringency. These results indicate that the short GCN repetitive sequences, which we have named ala, are found within transcribed regions of the Drosophila genome. These Drosophila genes containing the ala repeat do not show significant sequence similarity to any presently known gene; we have named these novel genes ala-A, ala-B, and ala-D. The cDNA clone from gene ala-B was named ala-E6.Key words: Drosophila melanogaster, repetitive DNA, GCN repeats, polyalanine encoded domain.

Structure and expression of histone H3.3 genes in Drosophila melanogaster and Drosophila hydei

Genome ◽  
1995 ◽  
Vol 38 (3) ◽  
pp. 586-600 ◽  
Author(s):  
Anna S. Akhmanova ◽  
Petra C. T. Bindels ◽  
Jie Xu ◽  
Koos Miedema ◽  
Hannie Kremer ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Sequence Similarity ◽  
Expression Patterns ◽  
Alternative Polyadenylation ◽  
Cdna Clones ◽  
Drosophila Hydei ◽  
Histone H3.3 ◽  
Somatic Tissues ◽  
Polyadenylation Sites ◽  
Limited Sequence

We demonstrate that in Drosophila melanogaster the histone H3.3 replacement variant is encoded by two genes, H3.3A and H3.3B. We have isolated cDNA clones for H3.3A and cDNA and genomic clones for H3.3B. The genes encode exactly the same protein but are widely divergent in their untranslated regions (UTR). Both genes are expressed in embryos and adults; they are expressed in the gonads as well as in somatic tissues of the flies. However, only one of them, H3.3A, shows strong testes expression. The 3′ UTR of the H3.3A gene is relatively short (~250 nucleotides (nt)). H3.3B transcripts can be processed at several polyadenylation sites, the longest with a 3′ UTR of more than 1500 nt. The 3′ processing sites, preferentially used in the gonads and somatic tissues, are different. We have also isolated the Drosophila hydei homologues of the two H3.3 genes. They are quite similar to the D. melanogaster genes in their expression patterns. However, in contrast to their vertebrate counterparts, which are highly conserved in their noncoding regions, the Drosophila genes display only limited sequence similarity in these regions.Key words: H3.3 histone variant, Drosophila, sequence comparison, alternative polyadenylation, testis expression.

scholarly journals The Gene Search System: A Method for Efficient Detection and Rapid Molecular Identification of Genes in Drosophila melanogaster

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 725-737 ◽  
Author(s):  
Gakuta Toba ◽  
Takashi Ohsako ◽  
Naomasa Miyata ◽  
Tsuyoshi Ohtsuka ◽  
Ki-Hyeon Seong ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Core Promoter ◽  
Sequence Similarity ◽  
Direct Sequencing ◽  
P Element ◽  
Drosophila Genome ◽  
Coding Region ◽  
New Genes ◽  
Gene Search ◽  
Efficient Detection

Abstract We have constructed a P-element-based gene search vector for efficient detection of genes in Drosophila melanogaster. The vector contains two copies of the upstream activating sequence (UAS) enhancer adjacent to a core promoter, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the basis of phenotypic changes caused by GAL4-dependent forced expression of vector-flanking DNA, and the transcripts were identified with reverse transcriptase PCR (RT-PCR) using the vector-specific primer and followed by direct sequencing. The system had a greater sensitivity than those already in use for gain-of-function screening: 64% of the vector insertion lines (394/613) showed phenotypes with forced expression of vector-flanking DNA, such as lethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% matched the sequences of cloned genes, and 18% matched reported expressed sequence tags (ESTs). Of the insertions in cloned genes, 83% were upstream of the protein-coding region. We discovered two new genes that showed sequence similarity to human genes, Ras-related protein 2 and microsomal glutathione S-transferase. The system can be useful as a tool for the functional mapping of the Drosophila genome.

scholarly journals Structure of transcripts from the homeotic Antennapedia gene of Drosophila melanogaster: two promoters control the major protein-coding region.

1986 ◽  
Vol 6 (12) ◽  
pp. 4676-4689 ◽  
Author(s):  
A Laughon ◽  
A M Boulet ◽  
J R Bermingham ◽  
R A Laymon ◽  
M P Scott
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Polyadenylation ◽  
Transcription Unit ◽  
Cdna Clones ◽  
Major Protein ◽  
Coding Region ◽  
Developmentally Regulated ◽  
Protein Coding ◽  
Reading Frame ◽  
C Terminus

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster regulates segmental identity in the thorax. Loss of Antp function results in altered development of the embryonic thoracic segments or can cause legs to be transformed into antennae. Certain combinations of Antp recessive lethal alleles complement to permit normal development. The structure of the Antp gene, analyzed by sequencing cDNA clones and exons and by transcript mapping, revealed some of the basis for its genetic complexity. It has two promoters governing two nested transcription units, one unit 36 and one 103 kilobase pairs (kb) long. Both units incorporated the same protein-coding exons, all of which are located in the 3'-most 13 kb of the gene. The two promoters resulted in the attachment of either of two long noncoding leader sequences (1.5 and 1.7 kb) to a 1.1-kb open reading frame. Both transcription units used the same pair of alternative polyadenylation sites 1.4 kb apart; the choice of sites was developmentally regulated. Some of the mutations that disrupt the larger transcription unit complemented a mutation affecting the smaller one. Dominant mutations that transform antennae into legs split the gene but left the coding exons intact. The encoded protein has unusually long runs of glutamine and a homeodomain near the C terminus.

Assembly of the Alu domain of the signal recognition particle (SRP): dimerization of the two protein components is required for efficient binding to SRP RNA

1990 ◽  
Vol 10 (2) ◽  
pp. 777-784
Author(s):  
K Strub ◽  
P Walter
Keyword(s):  
Cdna Clone ◽  
Repetitive Sequences ◽  
Signal Recognition Particle ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Cdna Clones ◽  
Functional Domain ◽  
Signal Recognition ◽  
Srp Rna

The signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, plays an essential role in targeting secretory proteins to the rough endoplasmic reticulum membrane. In addition to the targeting function, SRP contains an elongation arrest or pausing function. This function is carried out by the Alu domain, which consists of two proteins, SRP9 and SRP14, and the portion of SRP (7SL) RNA which is homologous to the Alu family of repetitive sequences. To study the assembly pathway of the components in the Alu domain, we have isolated a cDNA clone of SRP9, in addition to a previously obtained cDNA clone of SRP14. We show that neither SRP9 nor SRP14 alone interacts specifically with SRP RNA. Rather, the presence of both proteins is required for the formation of a stable RNA-protein complex. Furthermore, heterodimerization of SRP9 and SRP14 occurs in the absence of SRP RNA. Since a partially reconstituted SRP lacking SRP9 and SRP14 [SRP(-9/14)] is deficient in the elongation arrest function, it follows from our results that both proteins are required to assemble a functional domain. In addition, SRP9 and SRP14 synthesized in vitro from synthetic mRNAs derived from their cDNA clones restore elongation arrest activity to SRP(-9/14).

scholarly journals A highly conserved mouse gene with a propensity to form pseudogenes in mammals.

1988 ◽  
Vol 8 (7) ◽  
pp. 2797-2803 ◽  
Author(s):  
D L Heller ◽  
K M Gianola ◽  
L A Leinwand
Keyword(s):  
Dna Sequence ◽  
Cdna Clone ◽  
Restriction Analysis ◽  
In Vitro Transcription ◽  
Open Reading Frame ◽  
Cdna Clones ◽  
Reading Frame ◽  
Mouse Cdna ◽  
Processed Pseudogenes

A mouse cDNA clone corresponding to an abundantly transcribed poly(A)+ mRNA was found to be represented by 200 copies in mammalian genomes. To understand the origin and nature of this sequence family, we studied two genomic members and two cDNA clones from mouse liver. The DNA sequence of the coding strand of a full-length cDNA clone was shown to have an open reading frame capable of encoding a 25-kilodalton polypeptide that has not been previously described. In vitro transcription-translation experiments verified the presence of an open reading frame encoding a protein of the predicted size. Restriction analysis of genomic DNA and DNA sequence analysis of genomic clones indicated that many of the 200 members of this family represent processed pseudogenes, with one or a small number of active structural genes. The vast majority of the genomic copies are heterogeneous in length, truncated at their 5' ends with respect to the mRNA, and do not appear to have intervening sequences. Two distinct genomic members of this family were sequenced and found to represent incomplete copies of the mRNA. Both are 5' truncated at slightly different points with respect to the mRNA. Both pseudogenes have multiple base changes, insertions, and deletions relative to the mRNA, and one of them encodes the poly(A) tail of the mRNA. The expression of this gene family is highest in rapidly dividing cells such as early mouse embryos and testis, but was seen in all tissues tested. This gene shows extremely high sequence conservation, extending to chicken, amphibian, and nematode genomes. Surprisingly, the gene appears to exist in only one copy in these organisms.

scholarly journals Expression of a cDNA clone encoding the haem-binding domain of Chlorella nitrate reductase

1991 ◽  
Vol 278 (1) ◽  
pp. 203-209 ◽  
Author(s):  
A C Cannons ◽  
N Iida ◽  
L P Solomonson
Keyword(s):  
Cdna Clone ◽  
Spinacia Oleracea ◽  
Sequence Similarity ◽  
Cytochrome B5 ◽  
Binding Domain ◽  
Higher Plant ◽  
Expression Library ◽  
Reading Frame ◽  
Unicellular Green Alga ◽  
Partial Cdna Clone

A partial cDNA clone coding for the haem-binding domain of NADH:nitrate reductase (EC 1.6.6.1) (NR) from the unicellular green alga Chlorella vulgaris has been isolated, sequenced and expressed. A 1.2 kb cDNA (pCVNR1) was isolated from a lambda gt11 expression library produced from polyadenylated RNA extracted from nitrate-grown Chlorella cells. pCVNR1 hybridized to a 3.5 kb mRNA transcript that was nitrate-inducible and absent from ammonium-grown cells. The entire sequence of pCVNR1 was obtained and found to have a single uninterrupted reading frame. The derived amino acid sequence of 318 amino acids has a 45-50% similarity to higher-plant NRs, including Arabidopsis thaliana, spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum). A comparison with the putative domain structure of higher-plant nitrate reductases suggested that this sequence contains the complete haem-binding domain, approximately one-third of the Mo-pterin domain and no FAD-binding domain. A 32% sequence similarity is evident when comparing the Chlorella NR haem domain with that of calf cytochrome b5. Expression of pCVNR1 in a pET vector synthesized a 35 kDa protein that was antigenic to anti-(Chlorella NR) antibody. The spectral properties of this protein (reduced and oxidized) in the 400-600 nm region are identical with those of native Chlorella NR and indicate that haem is associated with the protein.

Structure of transcripts from the homeotic Antennapedia gene of Drosophila melanogaster: two promoters control the major protein-coding region

1986 ◽  
Vol 6 (12) ◽  
pp. 4676-4689
Author(s):  
A Laughon ◽  
A M Boulet ◽  
J R Bermingham ◽  
R A Laymon ◽  
M P Scott
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Polyadenylation ◽  
Transcription Unit ◽  
Cdna Clones ◽  
Major Protein ◽  
Coding Region ◽  
Developmentally Regulated ◽  
Protein Coding ◽  
Reading Frame ◽  
C Terminus

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster regulates segmental identity in the thorax. Loss of Antp function results in altered development of the embryonic thoracic segments or can cause legs to be transformed into antennae. Certain combinations of Antp recessive lethal alleles complement to permit normal development. The structure of the Antp gene, analyzed by sequencing cDNA clones and exons and by transcript mapping, revealed some of the basis for its genetic complexity. It has two promoters governing two nested transcription units, one unit 36 and one 103 kilobase pairs (kb) long. Both units incorporated the same protein-coding exons, all of which are located in the 3'-most 13 kb of the gene. The two promoters resulted in the attachment of either of two long noncoding leader sequences (1.5 and 1.7 kb) to a 1.1-kb open reading frame. Both transcription units used the same pair of alternative polyadenylation sites 1.4 kb apart; the choice of sites was developmentally regulated. Some of the mutations that disrupt the larger transcription unit complemented a mutation affecting the smaller one. Dominant mutations that transform antennae into legs split the gene but left the coding exons intact. The encoded protein has unusually long runs of glutamine and a homeodomain near the C terminus.

A highly conserved mouse gene with a propensity to form pseudogenes in mammals

1988 ◽  
Vol 8 (7) ◽  
pp. 2797-2803
Author(s):  
D L Heller ◽  
K M Gianola ◽  
L A Leinwand
Keyword(s):  
Dna Sequence ◽  
Cdna Clone ◽  
Restriction Analysis ◽  
In Vitro Transcription ◽  
Open Reading Frame ◽  
Cdna Clones ◽  
Reading Frame ◽  
Mouse Cdna ◽  
Processed Pseudogenes

A mouse cDNA clone corresponding to an abundantly transcribed poly(A)+ mRNA was found to be represented by 200 copies in mammalian genomes. To understand the origin and nature of this sequence family, we studied two genomic members and two cDNA clones from mouse liver. The DNA sequence of the coding strand of a full-length cDNA clone was shown to have an open reading frame capable of encoding a 25-kilodalton polypeptide that has not been previously described. In vitro transcription-translation experiments verified the presence of an open reading frame encoding a protein of the predicted size. Restriction analysis of genomic DNA and DNA sequence analysis of genomic clones indicated that many of the 200 members of this family represent processed pseudogenes, with one or a small number of active structural genes. The vast majority of the genomic copies are heterogeneous in length, truncated at their 5' ends with respect to the mRNA, and do not appear to have intervening sequences. Two distinct genomic members of this family were sequenced and found to represent incomplete copies of the mRNA. Both are 5' truncated at slightly different points with respect to the mRNA. Both pseudogenes have multiple base changes, insertions, and deletions relative to the mRNA, and one of them encodes the poly(A) tail of the mRNA. The expression of this gene family is highest in rapidly dividing cells such as early mouse embryos and testis, but was seen in all tissues tested. This gene shows extremely high sequence conservation, extending to chicken, amphibian, and nematode genomes. Surprisingly, the gene appears to exist in only one copy in these organisms.

A cDNA clone encoding an IgE-binding protein from Brassica anther has significant sequence similarity to Ca2+-binding proteins

1995 ◽  
Vol 29 (6) ◽  
pp. 1157-1165 ◽  
Author(s):  
Kinya Toriyama ◽  
Takashi Okada ◽  
Masao Watanabe ◽  
Takeshi Ide ◽  
Tsuneo Ashida ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Binding Proteins ◽  
Binding Protein ◽  
Sequence Similarity ◽  
Significant Sequence Similarity ◽  
Ige Binding

scholarly journals Developmental and metabolic regulation of the Drosophila melanogaster 3-hydroxy-3-methylglutaryl coenzyme A reductase.

1988 ◽  
Vol 8 (7) ◽  
pp. 2713-2721 ◽  
Author(s):  
F B Gertler ◽  
C Y Chiu ◽  
L Richter-Mann ◽  
D J Chin
Keyword(s):  
Drosophila Melanogaster ◽  
Metabolic Regulation ◽  
Coenzyme A ◽  
Terminal Region ◽  
Cdna Clones ◽  
Posttranslational Regulation ◽  
Hmg Coa Reductase ◽  
Reading Frame ◽  
Reductase Gene ◽  
Coa Reductase

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in Drosophila melanogaster synthesizes mevalonate for the production of nonsterol isoprenoids, which are essential for growth and differentiation. To understand the regulation and developmental role of HMG CoA reductase, we cloned the D. melanogaster HMG CoA reductase gene. The nucleotide sequence of the Drosophila HMG CoA reductase was determined from genomic and cDNA clones. A 2,748-base-pair open reading frame encoded a polypeptide of 916 amino acids (Mr, 98,165) that was similar to the hamster HMG CoA reductase. The C-terminal region had 56% identical residues and the N-terminal region had 7 potential transmembrane domains with 32 to 60% identical residues. In hamster HMG CoA reductase, the membrane regions were essential for posttranslational regulation. Since the Drosophila enzyme is not regulated by sterols, the strong N-terminal similarity was surprising. Two HMG CoA reductase mRNA transcripts, approximately 3.2 and 4 kilobases, were differentially expressed throughout Drosophila development. Mevalonate-fed Schneider cells showed a parallel reduction of both enzyme activity and abundance of the 4-kilobase mRNA transcript.

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