Analysis of testis - expressed Stellate genes promoter region in D. melanogaster
Here we present our analysis of a promoter-proximal region of Stellate genes expressed in D. melanogaster germline. We determined the minimal core promoter of Stellate genes using the series of deletion transgene constructs. We analyzed this region by gel shift assay and revealed three E-box site...
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| Cite this: | Analysis of testis - expressed Stellate genes promoter region in D. melanogaster / O.M. Olenkina, L.V. Olenina, S.A. Lavrov, M.S. Klenov, V.A. Gvozdev // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2008. — Т. 4. — С. 285-289. — Бібліогр.: 11 назв. — англ. |
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nasplib_isofts_kiev_ua-123456789-1779002025-02-23T18:22:55Z Analysis of testis - expressed Stellate genes promoter region in D. melanogaster Olenkina, O.M. Olenina, L.V. Lavrov, S.A. Klenov, M.S. Gvozdev, V.A. Механізми взаємодії та експресії генетичних систем Here we present our analysis of a promoter-proximal region of Stellate genes expressed in D. melanogaster germline. We determined the minimal core promoter of Stellate genes using the series of deletion transgene constructs. We analyzed this region by gel shift assay and revealed three E-box sites which bound to the protein factor from testis nuclear extract. Здесь мы представляем наш анализ промоторной области генов Stellate, экспрессирующихся в герминальных клетках D.melanogaster. Мы определили минимальный коровый промотор с помощью делеционных трансгенных конструкций. Мы проанализировали его с помощью гель-шифта и выявили три Е-бокса, связывающихся с белковым фактором из ядерного экстракта семенников. Тут ми представляємо наш аналіз промоторної ділянки генів Stellate, що експресується в гермінальних клітинах D.melanogaster. Ми визначили мінімальний коровий промотор за допомогою делецiонних трансгенних конструкцій. Ми проаналізували його за допомогою гель-шифту та виявили три дiлянки так званих Е- боксiв, що зв'язуються з білковим фактором з ядерного екстракту семенникiв. 2008 Article Analysis of testis - expressed Stellate genes promoter region in D. melanogaster / O.M. Olenkina, L.V. Olenina, S.A. Lavrov, M.S. Klenov, V.A. Gvozdev // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2008. — Т. 4. — С. 285-289. — Бібліогр.: 11 назв. — англ. 2219-3782 https://nasplib.isofts.kiev.ua/handle/123456789/177900 en Фактори експериментальної еволюції організмів application/pdf Інститут молекулярної біології і генетики НАН України |
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Механізми взаємодії та експресії генетичних систем Механізми взаємодії та експресії генетичних систем |
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Механізми взаємодії та експресії генетичних систем Механізми взаємодії та експресії генетичних систем Olenkina, O.M. Olenina, L.V. Lavrov, S.A. Klenov, M.S. Gvozdev, V.A. Analysis of testis - expressed Stellate genes promoter region in D. melanogaster Фактори експериментальної еволюції організмів |
| description |
Here we present our analysis of a promoter-proximal region of Stellate genes
expressed in D. melanogaster germline. We determined the minimal core promoter of Stellate
genes using the series of deletion transgene constructs. We analyzed this region by gel shift
assay and revealed three E-box sites which bound to the protein factor from testis nuclear
extract. |
| format |
Article |
| author |
Olenkina, O.M. Olenina, L.V. Lavrov, S.A. Klenov, M.S. Gvozdev, V.A. |
| author_facet |
Olenkina, O.M. Olenina, L.V. Lavrov, S.A. Klenov, M.S. Gvozdev, V.A. |
| author_sort |
Olenkina, O.M. |
| title |
Analysis of testis - expressed Stellate genes promoter region in D. melanogaster |
| title_short |
Analysis of testis - expressed Stellate genes promoter region in D. melanogaster |
| title_full |
Analysis of testis - expressed Stellate genes promoter region in D. melanogaster |
| title_fullStr |
Analysis of testis - expressed Stellate genes promoter region in D. melanogaster |
| title_full_unstemmed |
Analysis of testis - expressed Stellate genes promoter region in D. melanogaster |
| title_sort |
analysis of testis - expressed stellate genes promoter region in d. melanogaster |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2008 |
| topic_facet |
Механізми взаємодії та експресії генетичних систем |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/177900 |
| citation_txt |
Analysis of testis - expressed Stellate genes promoter region in D. melanogaster / O.M. Olenkina, L.V. Olenina, S.A. Lavrov, M.S. Klenov, V.A. Gvozdev // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2008. — Т. 4. — С. 285-289. — Бібліогр.: 11 назв. — англ. |
| series |
Фактори експериментальної еволюції організмів |
| work_keys_str_mv |
AT olenkinaom analysisoftestisexpressedstellategenespromoterregionindmelanogaster AT oleninalv analysisoftestisexpressedstellategenespromoterregionindmelanogaster AT lavrovsa analysisoftestisexpressedstellategenespromoterregionindmelanogaster AT klenovms analysisoftestisexpressedstellategenespromoterregionindmelanogaster AT gvozdevva analysisoftestisexpressedstellategenespromoterregionindmelanogaster |
| first_indexed |
2025-11-24T09:23:52Z |
| last_indexed |
2025-11-24T09:23:52Z |
| _version_ |
1849663141995085824 |
| fulltext |
285
10. Sohn J., Choi E., Kang H., Rhee J., Agaphonov M., Ter-Avanesyan M., Rhee S. A
dominant selection system designed for copy-number-controlled gene integration in
Hansenula polymorpha DL-1 // Appl. Microbiol. Biotechnol.- 1999.- vol. 51, P.800–
807.
11. Veenhuis M., Hoogkamer-Te Niet M.C., Middelhoven W.J. Biogenesis and metabolic
significance of microbodies in urate-utilizing yeasts // Antonie Van Leeuwenhoek-
1985.-vol. 51, P.33-43.
12. Li J., Chen Z., Hou L., Fan H., Weng S., Xu C., Ren J., Li B., Chen W. High-level
expression, purification, and characterization of non-tagged Aspergillus flavus urate
oxidase in Escherichia coli // Protein Expr. Purif.- 2006.- vol. 49, P.55-59.
13. Leplatois P., Douarin B., Loison G. High-level production of a peroxisomal enzyme:
Aspergillus flaws uricase accumulates intracellularly and is active in Saccharomyces
cerevisiae // Gene– 1992.- vol. 122, P.139-145.
Abstract
Strains of Hansenula polymorpha overproducing own uricase (UOX) have been
constructed. Uricase gene under the control of the strong H. polymorpha alcohol oxidase
promoter was multicopy integrated to the recipient strain H. polymorpha С-105 (gcr1 catX).
The recombinant producer characterized by a forty-fold increased UOX enzyme activity (up
to 3.36 U ml-1 in cell-free extract) compared to the initial strain.
Сконструированы штаммы Hansenula polymorpha с усиленной экспрессией
собственной уриказы. Ген уриказы под контролем сильного промотора гена
алкогольоксидазы H. polymorpha введён в геном реципиентного штамма H. polymorpha
С-105 (gcr1 catX). Рекомбинантный штамм характеризировался сорокакратным
увеличением активности уриказы (3.36 МЕ мл-1 в бесклеточных экстрактах) в
сравнении с исходным штаммом.
Сконструйовані штами Hansenula polymorpha з посиленою експресією власної
урікази. Ген урікази під контролем сильного промотора алкогольоксидази H.
рolymorpha було введено в геном реципієнтного штаму H. polymorpha С-105 (gcr1
catX). Рекомбінантний штам характеризувався підвищенням активності урікази в 40
разів (3.36 МЕ мл-1 в безклітинних екстрактах) у порівнянні з вихідним штамом.
OLENKINA O.M.,OLENINA L. V., LAVROV S.A., KLENOV M. S., GVOZDEV V.A.
Department of Animal Molecular Genetics, Institute of Molecular Genetics,
Kurchatov sq., 2, Moscow, 123182, Russia; e-mail: olenkina@img.ras.ru
ANALYSIS OF TESTIS - EXPRESSED STELLATE GENES PROMOTER REGION
IN D. melanogaster
The first described case of a natural RNA-mediated silencing found to be necessary
for male fertility maintenance was discovered in Drosophila (Aravin et al., 2001). In wild-
type testes, hyperexpression of tandemly repeated X-linked Stellate genes is prevented by
homologous, bidirectionally transcribed Y-linked Suppressor of Stellate repeats (Su(Ste)), and
deletion of Su(Ste) leads to abnormalities of spermatogenesis (Palumbo et al., 1994). In the
Drosophila germline, selfish genetic elements, such as retrotransposons and repetitive
sequences, are suppressed by rasiRNAs (Vagin et al., 2006). However, a little is known about
classical transcription regulation of the Stellate genes. The expression of the Stellate genes is
restricted to D. melanogaster testes. The transcription start site was determined in (Livak,
286
1990) by primer extension experiments. Sequence in -30 region upstream transcription start
has shown no TATA-element similarity, so the Stellate gene promoter appears to be TATA-
less but initiator-containing one. As we have shown earlier (Aravin et al., 2001) a 134 bp-long
region containing Stellate initiator site is sufficient for high-level expression of transgene
reporter LacZ in testes of cry1 males. This result provides a strong evidence for a presence of
cis-regulatory sites within this region. In this article, we present our analysis of the promoter
region of the Stellate genes.
Materials and methods
Reporter construction design. For PCR amplification, a plasmid template containing 6
full-length Stellate genes was used. PCR amplifications of Stellate regulatory sequences with
primers, generating a duplex with 5’ overhangs containing sites for XbaI and BamHI, and
ligation in pCaSpeR-β-gal vector opened with XbaI and BamHI, were performed as in Aravin
et al., 2001.
Drosophila strains, transformation, and genetic crosses. Flies were reared on standard
medium at 25ºC. P-element-mediated germline transformation of Df(1)w67c23(2)y embryos was
performed according to standard protocol (Rubin and Spradling, 1982). The number of
insertions in genome was estimated by Southern blot. The strain with deletion of the bulk of
Su(Ste) repeats on the Y chromosome was cry1Y, described in Palumbo et al., 1994. To
produce males carrying the cry1Y chromosome, Df(1) w67c23(2)y females were crosses to
X/cry1BsYy+ males.
Testis nuclear extract preparation. It was prepared as in (Dignam et al., 1983) with
minor modifications.
Electromobility shift assay. The binding probes were terminally [32P]-labeled with T4
polynucleotide kinase. Testis nuclear extract 10-15 micrograms was incubated for 20 min at
room temperature with 0.3-0.6 pmol of labeled oligonucleotide. All binding reaction were
carried out in 20 mM HEPES pH7.6, 50-60 mM NaCl, 0.1 mM EDTA, 5 mM MgCl2, 1mM
DTT, 5% glycerol, 20 ng/μl poly(dI-dC), 0.1 ng/μl heparin in total volume of 15 μl. DNA-
protein complexes were resolved by 5% non-denaturing polyacrylamide gel electrophoresis in
0.75xTris-borate-EDTA buffer with 2.5% glycerol at 8ºC.
Results and discussion
Determination of 5’ border of proximal promoter region of Stellate genes. General
approach taken to determine upstream borders of gene transcription-control regions involves
creating a set of 5’-deleted reporter constructs. It was previously shown that lacZ reporters
driven by 5’ Stellate fragments carrying the Ste225-lacZ and Ste131-lacZ constructs were
sufficient to provide high-level expression of β-galactosidase in cry1Y males testes (Aravin et
al., 2001). 134 bp 5’-fragment of Stellate heterochromatin gene contains 33 bp of 5’-
transcribed region from ATG start codon, and promoter–proximal region lying 101 bp
upstream of the start site. To determine whether the 134 bp fragment can be account as
minimal promoter region of the Stellate genes, we constructed two additional 5’-deletion
mutant constructs of Stellate promoter-proximal region Ste63-lacZ and Ste44-lacZ with lacZ-
reporter gene, and established the transgenic fly lines by P-element-mediated germline
transformation. Both mutant constructs also contained 33 bp of 5’-transcribed region of
Stellate gene sequence downstream from the ATG start codon fused with lacZ gene and
untranscribed 5’ upstream fragments of Stellate heterochromatin gene of 30 bp or 11 bp,
respectively. Analysis of β-galactosidase activity in dissected testes from the cry1 males
revealed that β-galactosidase expression of the Ste63-lacZ was significantly weaker than that
of the Ste131-lacZ (fig.1).
287
Figure 1. Activity of β-galactosidase expressed in testes of D. melanogaster under
control of the Stellate promoter sequences of different sizes.
The Ste44-lacZ construct did not show β-galactosidase activity and did not differ
markedly from control fly lines, which were used for P-element transformation. In our
experiments, only flies bearing the Ste131-lacZ construct allowed high-level expression of the
lacZ reporter in testes. As a result, we consider the fragment of 134 bp (-101 bp upstream and
+33 bp downstream transcription start site) to be a minimal promoter region of the Stellate
genes, which is sufficient for their correct testes-specific transcription. We suppose that the
short 71 bp fragment (from -101 bp to -30 bp) shared by the Ste131-lacZ and the Ste63-lacZ
constructs contains a cis-acting protein-binding control element, or a few elements, which
regulate Stellate gene transcription in testes.
Identification of cis-acting elements within Stellate promoter. To solve the question
which cis-acting elements for testes-specific transcription are present in Stellate promoter,
electromobility shift assay was performed. We used four 5’-radiolabeled oligonucleotides in
27-37 bp encompassing the minimal promoter of 134 bp and named Ste1, Ste2, Ste3 and Ste4
as probes. We observed three specific DNA-protein complexes formed with Ste1, Ste2, and
Ste4 oligonucleotides, respectively, after their incubation with testis nuclear extract (fig.2). In
all of these cases, 50-fold excess of unlabeled specific oligonucleotide was able to compete
for binding, whereas nonspecific was not. The signal intensity of binding complexes
decreased reproducibly in next array: Ste2 > Ste4 > Ste1. Within Ste2 we found palindrome
hexanucleotide sequence CACGTG. Sequence CANNTG is well known as transcription
control element named E-box (Atchley et al., 1997; Ledent and Vervoort, 2001). We aligned
Ste1 and Ste4 oligonucleotides using Ste2 as a template and found that both of them
contained degenerated E-box sequences: CATCTG and CAAGTG, respectively.
Figure 2. Electromobility shift assay.1- without extract; 2, 3, 4- with testis nuclear
extract; 3- in presence of 50-fold excess of unlabeled specific oligonucleotide; 4- in presence
of 50-fold excess of unlabeled nonspecific oligonucleotide.
0
0,2
0,4
0,6
0,8
1
1Ste63-lacZ Ste131-lacZ Ste44-lacZ
A
lac Z
288
To verify if this testes-specific protein factor binds just E-box in the Ste2 sequence,
we performed gel shift assay with mutated Ste2 oligonucleotide, Ste2mut, where E-box
CACGTG sequence was replaced by GGCTAT. Using Ste2mut as radiolabeled probe with
testis nuclear extract abolished the gel shift (data are not shown). These observations suggest
that the gel shift complex of Ste2 oligonucleotide is determined by the E-box motif. We also
have noted that all three complexes run shifted probes in gel nearly with the same mobility.
To confirm that all three oligonucleotides Ste1, Ste2 and Ste4 bind the same factor, we
designed cross-competition assay. We assumed that the unlabeled Ste2 oligonucleotide
containing the perfect E-box would compete with all E-box-containing oligonucleotides for
the E-box binding factor, whereas Ste2mut would not. Actually, we observed that the 70-fold
molar excess of unlabeled Ste2 competed effectively for complex formation with all three
radiolabeled oligonucleotides, however the same excess of unlabeled Ste2mut did not affect
the binding (fig. 3). Therefore, we determined three cis-acting motifs within minimal Stellate
promoter to be E-boxes and detected the single protein factor from testis nuclear extract
interacting with them in vitro in our experiments.
Figure 3. Cross-competition shift assay. Testis nuclear extract was incubated with
oligonucleotide probes. 1- without competitors; 2- in presence of 70-fold excess of unlabeled
Ste2 oligonucleotide; 3- in presence of 70-fold excess of unlabeled Ste2mut oligonucleotide.
#-unspecific binding.
Discussion. The most of genes are transcribed during the organism life cycle
according to the environmental conditions and in certain cell types and organs. The changing
set of transcription factors provides control of place, time, and level of transcription for every
particular gene. The composition of transcription factors binding sites (cis-regulatory regions)
near a gene is the major determinants of its expression. The binding sites comprise a small
part of nucleotides within promoter region. This fraction ranges from 10-20% within well-
studied regulatory regions (Wray et al., 2003). Some genes analyzed to date, expressing only
during Drosophila spermatogenesis, have extremely short regulatory regions located near
basal promoter (Santel, 2000; Blumer et al., 2002). Here we determined a fragment of 134 bp
as a minimal promoter region of the Stellate genes, which is sufficient for their correct testes-
specific transcription. We identified three cis-regulatory sites within this minimal Stellate
promoter in vitro. These cis-regulatory sites are known as E-boxes. The perfect E-box
CACGTG is located from -47 bp to -42 bp upstream transcription start site of the Stellate
gene. It resides within the promoter fragment of 71 bp which has been shown above to be
responsible for the high-level expression of the reporter gene. E-box regulatory sites have
been identified in a lot of promoter and enhancer elements. The E-box motif is known as
cognate recognition sequence for basic helix-loop-helix (bHLH) superfamily of
transcriptional regulatory proteins that are found in organisms ranging from yeast to humans.
289
According to our data, we can expect that the protein which binds to the E-boxes in our
experiments also belongs to the bHLH superfamily.
Conclusions
We determined fragment of 134 bp (-101 bp upstream and +33 bp downstream
transcription start site) to be minimal promoter region of the testis-expressed Stellate genes in
D. melanogaster. We also identified three cis-regulatory sites within it to be E-boxes and
showed that all of them interacted with the same DNA-binding factor from testis nuclear
extract.
References
1. Aravin A.A. et al. Double-stranded RNA-mediated silencing of genomic tandem repeats
and transposable elements in the D. melanogaster germline//Curr.Biol.-2001.-v.11(13).-
P.1017-1027.
2. Palumbo G et al. Genetic analysis of Stellate elements of Drosophila
melanogaster//Genetics.-1994.-v.138.-P.1181-1197.
3. Vagin V.V. et al. A distinct small RNA pathway silences selfish genetic elements in the
germline//Science.-2000.-v.313(5785).-P.320-324.
4. Livak K. Detailed structure of the Drosophila melanogaster stellate genes and their
transcripts//Genetics.-1990.-v. 124(2).-P.303-316.
5. Rubin G. and Spradling A. Genetic transformation of Drosophila with transposable
element vectors//Science.-1982.-v.218.-P.348-353.
6. Dignam J.D. et al. Accurate transcription initiation by RNA polymerase II in a soluble
extract from isolated mammalian nuclei//Nucleic Acid Res.-1983.-v.11.-P.1475-1489.
7. Atchley W. and Fitch W. A natural classification of the basic helix-loop-helix class of
transcription factors//Proc. Natl. Acad. Sci. U S A.-1997.-v.94(10).-P.5172-5176.
8. Ledent V. and Vervoort M. The basic helix-loop-helix protein family: comparative
genomics and phylogenetic analysis//Genome Res.-2001.-v.11(5).-P.754-770.
9. Wray GA et al. The evolution of transcriptional regulation in eukaryotes//Mol. Biol. Evol.-
2003.-v 20(9).-P.1377-1419.
10. Santel A et al. The initiator element of the Drosophila β2tubulin gene core promoter
contributes to gene expression in vivo but not required for male germline specific
expression//Nucleic Acid Res.-2000.-v.28.-P.1439-1446.
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Abstract
Here we present our analysis of a promoter-proximal region of Stellate genes
expressed in D. melanogaster germline. We determined the minimal core promoter of Stellate
genes using the series of deletion transgene constructs. We analyzed this region by gel shift
assay and revealed three E-box sites which bound to the protein factor from testis nuclear
extract.
Здесь мы представляем наш анализ промоторной области генов Stellate,
экспрессирующихся в герминальных клетках D.melanogaster. Мы определили
минимальный коровый промотор с помощью делеционных трансгенных конструкций.
Мы проанализировали его с помощью гель-шифта и выявили три Е-бокса,
связывающихся с белковым фактором из ядерного экстракта семенников.
Тут ми представляємо наш аналіз промоторної ділянки генів Stellate, що
експресується в гермінальних клітинах D.melanogaster. Ми визначили мінімальний
коровий промотор за допомогою делецiонних трансгенних конструкцій. Ми
проаналізували його за допомогою гель-шифту та виявили три дiлянки так званих Е-
боксiв, що зв'язуються з білковим фактором з ядерного екстракту семенникiв.
|