|
http://www.abbs.info e-mail:[email protected] ISSN 0582-9879
ACTA BIOCHIMICA et
BIOPHYSICA SINICA 2003, 35(7): 597–600
CN 31-1300/Q |
(Morgan-Tan International
Center for Life Sciences, Institute of Developmental Biology and Molecular
Medicine, School of Life Science, Fudan University, Shanghai 200433, China)
More and more genes have been discovered through genomic sequencing. However, only a few of them have been functionally characterized in detail. The sequencing of human genome has revealed about 35 000－45 000 protein-coding genes, while only around 2000 genes have known functions[1,2]. A significant challenge for the post genome era is therefore to understand the functions of the newly sequenced genes. Unfortunately, so far there is no efficient system to provide functional clues for human genes in large scale.
Because of its powerful genetics and relatively low cost, Drosophila melanogaster is a very popular model organism in developmental biology studies[3]. The evolutionary conserved biological processes, as well as the participating genes shared between Drosophila and human， implied that Drosophila could be very helpful for studying human genes[4,5].
In this report, we tested the feasibility of characterizing functions of human genes by overexpressing them in Drosophila. Ten human genes were investigated with the GAL4-UAS system[6,7]. One of these genes, the translation elongation factor 1α1 (EF1α-1), led to abnormal notum and rough eye phenotypes when overexpressed with several GAL4 lines. These results suggested that overexpressing human genes in Drosophila could be used as a primary functional screen.
.
1 Materials and Methods
1.1 Plasmid construction
Human cDNAs were randomly picked from a human brain cDNA library and subcloned into pUAST vector with standard protocol[8, 9]. After sequencing, full-length clones were selected for DNA preparation and successive microinjection.
1.2 Stocks and culture conditions
The chromosomes and genetic markers used were described by Lindsley et al.[10]. All flies were reared on standard cornmeal-sucrose-yeast medium (http://flystocks.bio.indiana.edu/harvard-food.htm) at room temperature, unless specified otherwise.
Lines used for
mapping and balancing are FM7c/FM6 and yw; Adv/CyO; Sb/TM6B.
GAL4 lines are shown in Table 1.
1.3 Establishment of transgenic flies
Transgenic
plasmids were dissolved in injection buffer (5 mmol/L KCl, 0.1 mmol/L NaH
Survived F0 males were mated individually with three virgin w1118 females, while single F0 virgin female was mated individually with four w1118 males. Progenies from these crosses (F1 generation) with colored eye were used to establish transgenic lines by balancing with various balancer chromosomes, as shown in Fig.1.
1.4 Phenotype analysis
Virgin females
of each transgenic line were mated with males from different GAL4 lines,
respectively (Table 1). Progenies from these crosses were checked for visible
phenotypes under dissecting microscopes.
Table 1 GAL4 lines for use
|
Various GAL4
lines |
Expression
pattern |
|
actin-GAL4/CyO (II) |
Ubiquitous[11] |
|
pGMR-GAL4/pGMR-GAL4 (II) |
Eye-specific,
differenciated cells in the eye[12] |
|
ey-GAL4/CyO
(II) |
Eye-specific,
dividing cells in the eye[12] |
|
ptc-GAL4/ptc-GAL4 (II) |
Dorsal
mesothoracic disc that is relative of the wing, costal cells and wing veins[13] |
|
pnr-GAL4/TM3 (III) |
Mediodorsal
parts of thoracic and abdominal segments[14] |
|
Vg-GAL4/CyO (II) |
Wing-specific[15] |
2
Results
Ten human genes were microinjected into Drosophila, resulted in 54 different transgenic lines. Blast results (http://www.ncbi.nlm.nih.gov/blast) indicated that five of these genes encode novel proteins with unknown functions, while others encode known products (Table 2).
Table 2 Human genes transgenic fly balancing and mapping
|
Clone number |
Number of transgenic lines |
Gene products |
||
|
X# |
II# |
III# |
||
|
14-2(2.3 kb) |
1 |
1 |
4 |
Hypothetical protein |
|
1v-41(1 kb) |
|
|
3 |
Hypothetical protein |
|
2n-66(1.2 kb) |
|
5 |
5 |
Hypothetical protein |
|
2n-46(2.5 kb) |
|
4 |
2 |
Hypothetical protein |
|
1v-50(1 kb) |
|
2 |
2 |
HSPCa 163 protein |
|
1e-34(1.2 kb) |
|
2 |
3 |
QPRTb |
|
1v-27(1 kb) |
1 |
1 |
|
Ubiquitin B |
|
1v-06(1 kb) |
2 |
3 |
3 |
RPS2c |
|
1v-16(1.8 kb) |
|
4 |
2 |
EF1α-1[18, 19] |
|
1v-28(2 kb) |
|
3 |
1 |
SV2d protein |
#chromosome location; aundefined
genes expressed in CD34+ hematopoietic stem/progenitor cells; bquinolinate
phosphoribosyl transferase; cribosomal protein S2; dhomologous
to a family of proton cotransporters from bacteria and fungi and a related
family of glucose transporters found in mammals.
To test the
overexpression phenotype, several lines from each transgene were used to cross
with an array of 6 different GAL4 lines, respectively. Each cross has been
repeated for three times. One of the human genes, EF1α-1, gave
interesting phenotypes. When driven by pnr-GAL4, EF1α-1 expression led
to an abnormal notum with lack of bristles in the midline. Typical phenotypes
are shown in Fig.2. Weak alleles [Fig.2(B)] had slightly abnormal notums
compared to those of the control [Fig.2(A)], while notum of intermediate
alleles had a bristle gap in the middle line [Fig.2(C)]. The notum of strong
alleles were just looked as if to be splitted into two parts Fig.2(D)]. EF1α-1
could also lead to abnormality when expressed in the eye. By using pGMR-GAL4,
EF1α-1 overexpression made rough eyes. The number of the microchaetes among
the ommatidium altered [Fig.3(B)]. When the EF1α-1 transgenic fly lines
mated with ey-GAL4, Vg-GAL4, actin-GAL4 and ptc-GAL4 separately,
no obviously phenotypes were observed (data not shown)
Fig.2 Abnormal notum caused by EF1α-1 overexpression (crossed with pnr-GAL4 line)
The genotypes of each flies were: (A) pnr-GAL4/+. (B)
pnr-GAL4/+; EF1α-1/+ (chromosome II). (C) pnr-GAL4/ EF1α-1
(chromosome III). (D) pnr-GAL4/+; EF1α-1/+ (chromosome II). White
arrows indicate the bristle distribution on the middle of the notum.
Fig.3
Rough eyes caused by EF1α-1 overexpression (crossed with pGMR-GAL4 line)
The genotype were: (A) pGMR-GAL4/+. (B) pGMR-GAL4/+; EF1α-1/+. (C) The normal arrangement of the microchaetes. (D) Multiple microchaetes while (black frame). (E) Loss of the microchaetes among the ommatidium(white frame).
3 Discussion
3.1 Feasibility of screening human gene
functions by overexpression in Drosophila
Owing to its power in systematic screen and large amount of available GAL4 lines, GAL4-UAS system has been widely used in the genetic research[20]. In our study we tested the feasibility of annotating the human gene functions in Drosophila with this system by using ten human genes randomly selected. When driven by different GAL4 lines, one of these ten human genes showed detectable overexpression phenotypes. These results indicated the potential role of the GAL4-UAS system in large-scale screen of human gene functions.
Previous studies have shown that 2%－7% Drosophila genes can result in some phenotypes when overexpressed in a given spatial or temporal pattern[7, 12, 21－23]. Considering the homology between human and fly genomes (61%)[24], our previous estimation is that 5% human genes can show overexpression phenotypes in Drosophila. However, the result of this and other studies in our institute showed that about 10% human genes resulted in detectablee phenotypes when overexpressed in Drosophila. This may be because of the different GAL4 lines we used. Alternatively, it may reflect the biological differences between human and Drosophila.
Six different GAL4 lines were used in this pilot screen. One of them expresses ubiquitously, while others express in a tissue-specific manner. When driven by these GAL4 lines, functions of exogenous proteins could be tested in various tissues and stages in development. Moreover, most of the GAL4 lines we used expressing in the organs are easy to observe, which makes the screen more convenient.
In general,
overexpression in fly is suitable and convenient for primary annotation of
human gene function. The basic biological processes are highly conserved
between human and flies. Almost all important signaling pathways were first
identified in Drosophila or C. elegans. Previous studies have
also shown that when overexpression of human genes lead to abnormal phenotypes
in fly, it usually indicates that the exogenous genes are perturbing the
function of one or more potentially conserved signaling pathways. Further
investigation of the functions of these genes therefore, can be carried out
following the clues from Drosophila.
3.2 Potential role of EF1α-1 in
actin cytoskeleton
Some mutations affecting bristle morphology in Drosophila are caused by a perturbed actin cytoskeleton[25]. Also, signals mediated by the Notch pathway and the EGF signaling pathway can inhibit or promote macrochaetes development, respectively[26].
Overexpression
of EF1α-1 in yeast resulted in reduced budding and altered actin
distribution as well as cellular morphology[19]. Thus lack or twisted bristles [Fig.3(D)
and 3(E)], as well as their abnormal distribution on the notum [Fig.2(D)] of
the flies that overexpressing EF1α-1 may be a result of perturbed actin
cytoskeleton. However, this does not exclude the possibility that EF1α-1
overexpression could also lead to abnormal Notch or EGF signaling. Further
investigation of the functions of EF1α-1 may be focused on clarifying
the possible relationship between EF1α-1 and the pathways mentioned
above.
Acknowledgements We are grateful to Dr. XU Tian for suggestions and
critical reading. We also thank ZHU Huan-Hu, LIU Xu, DING Xu for transgenic
constructs, Dr. HAN Min, Dr. ZHUANG Yuan and other colleagues in Institute of
Developmental Biology and Molecular Medicine for discussion and technology
help.
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_________________________________
Received: April 15, 2003 ccepted: May 13, 2003
This work was supported by the grants from National Natural Science Foundation of China (No. 30030080) and Ph.D Acdemic Discipline Construction Program under Ministry of Education of China (No. 97024618)
*Corresponding author: Tel, 86-21-65643718;
Fax, 86-21-65643770; e-mail, [email protected]