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http://www.abbs.info e-mail:[email protected] ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(3): 306-310 CN 31-1300/Q |
Abstracts
of the 5th A-IMBN ConferencePartial Abstracts of the 5th Conference of the
Asia-Pacific
Infernational
Molecular Biology Network (A-IMBN)(Part Ⅱ)
The biosynthesis of aurodrosopterin in Drosophila melanogaster: Purification, cloning and characterization of dihydropterin deaminase
Chiyoung Ahn,
Jaekwang Kim, Sangick Park, Jeongbin Yim*
( School of
Biological Sciences, Seoul National University, Seoul 151-742, Korea, *e-mail:
[email protected] )
A dihydropterin deaminase, which catalyzes the
conversion of 7,8-dihydropterin to 7,8-dihydrolumazine, was discovered in Drosophila
melanogaster. In addition, 7,8-dihydrolumazine and
PDA{2-amino-4-oxo-6-acetyl-7,8-dihydro-3H,9H-pyrimido[4,5-b][1,4]diazepine}
could be converted into aurodrosopterin, an eye pigment, in the acidic
condition. Thus it has been assumed that a deamination reaction might be
involved in the aurodrosopterin synthesis. We purified the dihydropterin
deaminase to homogeneity by combination of gel permeation, phenyl-Sepharose
chromatography and chromatofocusing. The purified enzyme has a molecular weight
of 47 000, as analyzed by SDS-polyacrylamide gel electrophoresis. MALDI-TOF
mass spectrometry followed by peptide mass database searches identified the
purified protein as a product of CG18143. The product of CG18143 was highly
homologous to human and rat guanine deaminases, raising the possibility that it
encodes the dihydropterin deaminase. The product of CG18143 expressed in E.
coli had the enzyme activity, proving that it truly encodes the
dihydropterin deaminase. When the enzymatic reaction product was incubated with
PDA in acidic condition, the synthesis of aurodrosopterin was detected by thin
layer chromatography. This result suggests that the dihydropterin deaminase may
be directly involved in the biosynthesis of aurodrosopterin by converting
7,8-dihydropterin to 7,8-dihydrolumazine.
Toll-8 negatively regulates Dpp signaling in the developing Drosophila wing
Sangjoon Kim1, Changsoo Kim2, Seungbok Lee3, Jeongbin Yim4*
( 1National
Creative Research Initiative Center for Genetic Reprogramming, Institute of
Molecular Biology and Genetics, Seoul National University, 151-742, Korea,
e-mail: [email protected];
2Hanwha Chemical Research R&D Center,
305-345, Korea, e-mail: [email protected];
3National Creative Research Initiative
Center for Genetic Reprogramming, Institute of Molecular Biology and Genetics,
Seoul National University, 151-742, Korea, e-mail: [email protected];
4National Creative Research Initiative
Center for Genetic Reprogramming, Institute of Molecular Biology and Genetics,
Seoul National University, 151-742, Korea, *e-mail: [email protected] )
In Drosophila, Decapentaplegic (Dpp) acts as a key
regulator of wing development via induction of target genes, including spalt,
omb, and vgQ, in a concentration dependent manner. Dpp signaling appears to be
down-regulated by Brinker (Brk), a direct transcriptional repressor of Dpp
target genes. Here we show that Dpp signaling can be also negatively regulated
by Toll-8, a member of Toll receptor family. The protein contains a
Toll/Interleukin I Receptor (TIR) domain in the intracellular region and
leucine rich repeats (LRR) in the extracellular region. Expression of Toll-8 is
confined in the lateral region of the wing disc and is repressed by Dpp as is
Brk. We find that Toll-8 suppresses the phenotypic defects by ectopic
expression of Dpp, Tkv, or Mad in the wing. Ectopic expression of Toll-8 in the
medial region of the wing disc negatively regulates expression of a Dpp
downstream target gene, spalt, and this repression is restricted in the cells
that just abut mosaic clones over-expressing Toll-8. Interestingly, the
intracellular region of Toll-8 is not essential for spalt repression,
suggesting the cell non-autonomous mode of its action. We propose that a novel
cell-cell communication via Toll-8 may play an important role for the readout
of Dpp morphogen gradient during wing development.
Inhibition of human colorectal carcinoma cells by the citrus flavonoid tangeretin through cell-cycle G1-arrest and cyclin-dependent kinase suppression
Jen-Kun Lin1*, Min-Hsiung
Pan1, Shoei-Yn Lin-Shiau2
( 1Institutes
of Biochemistry and 2Toxicology, College of Medicine, Taiwan University,
Taipei, China; *e-mail: [email protected] )
Flavanoids are widespread in fruits and vegetables.
Citrus flavonoids such as tangeretin, nobiletin, apigenin and quercetin have a
broad spectrum of biological activity including anti-inflammatory and
anti-carcinogenic activities. Tangeretin (5,6,7,8,4’-pentamethoxyflavone) is
concentrated in the peel of citrus fruits. The results of DNA flow cytometric
analysis indicated that tangeretin blocked cell cycle progression at G1 phase
in colorectal carcinoma COLO 205 cells. The degree of phosphorylation of Rb was
decreased after 12 h and G1 arrest developed. The protein expression of cyclins
A, D1 and E reduced slightly under the same conditions. Immunocomplex kinase
experiments showed that tangeretin inhibited the activities of cyclin-dependent
kinases 2 (Cdk2) and 4 (Cdk4) in a dose-dependent manner in the cell-free
system.
Tangeretin also increased the content of the Cdk
inhibitor p21 protein and this effect correlated with the elevation in p53
levels. In addition, tangeretin also increased the level of the Cdk inhibitor
p27 protein within 18 h. These results suggested that tangeretin and some other
citrus flavonoids might either exert their growth-inhibitory effects and
anti-inflammatory and cancer chemopreventive actions through modulating the
activities of several key G1 regulatory proteins such as Cdk2 and Cdk4, or
mediate the increase of Cdk inhibitors p21 and p27.
Hua-Liang Jiang*, Jian-Hua
Shen, Xiao-Min Luo, Hong Liu, Xu Shen, Kai-Xian Chen
( Center for
Drug Discovery and Design, Shanghai Institute of Materia, Shanghai Institutes
of Biological Science, the Chinese Academy of Sciences, 294 Taiyuan Road,
Shanghai 200031, China, *e-mail: [email protected] )
The completion of the human genome suggests there are
30 000 to 40 000 genes and at least as many proteins. Many of these proteins
are potential targets for drug intervention to control human disease or injury,
and popular estimates are in the range from 2000 of 5000. Discovering lead
compounds that may be developed as new drugs against these new targets is an
exciting and stimulating challenge for the medicinal chemists and
pharmacologists. During the history of modern chemistry, organic chemists have
synthesized and isolated from natural sources more than 16 million organic
compounds. Among them less than 1% have been screened for drug discovery, about
7000 drugs have been found in this way. Therefore, we may believe that large
numbers of new drugs, at least many active compounds, are hiding in this
organic compound mine. However, how to mine this compound source is a hard task
of our medicinal chemists. Collecting all these 16 million compounds and
screening them randomly is unpractical, because it will cost billions of US
dollars for one target. While a new computational method——virtual screening
(VS) shows a dawning to solve this problem.
Virtual screening is a natural extension of molecular
docking or three-dimensional(3D) pharmacophore based database searching (PBDS),
capable of automatically evaluating very large databases of compounds. Two
strategies have been used in virtual screening: (1) using docking approach to
rank the databases if the 3D structures of the targets are available; (2) using
PBDS to identify potential hits from the databases if the 3D structures of the
targets are unknown. Normally, these two approaches are used synthetically,
because the later method can filter the compounds quickly and the former method
can evaluate ligand-receptor binding more accurately.
VS-by-docking is a new promising method. Doman et
al. compared the performance of random high throughput screening (HTS) and
molecular docking in searches for inhibitors of protein tyrosine phosphatase 1B
(PTP1B) ——a target for type 2 diabetes. The result indicated that docking
enriched the hit rate by 1700-fold over random screening. Li et al.
demonstrated the high efficiency of docking in ranking hits by their DockCrunch
project. They searched ACD-SC database (contains 1.1 million compounds at that
time, now it has about 2.2 million compounds) using docking approach against
the estrogen receptor. Among the 37 compounds selected from the virtual
screening, 21 compounds show activities less than 300 nmol/L, 14 compounds’
activities are less than 100 nmol/L and 2 are less than 10 nmol/L.
Recently, we have parallelized the well-known program
DOCK developed by Kuntz et al. in several types of supercomputers. This
speeds up the database screening dramatically. Employing this approach, we have
discovered a series of active compounds from the available databases MDL/ACD,
MDL/MDDR, ACD-SC and our own database CNPD (Chinese Natural Produce Database)
against several targets, such as tyrosine kinase, potassium ion channel, β-secretase,
MMPs and PPARγ. Targeting the ligand binding domain of
PPARγ, we screened ACD-SC (2.2 million compounds) using above parallelized
DOCK, 23 compounds with activity less than μmol/L were tackled, and 3 of them
show binding affinities higher than all the launched PPARγ
agonists at the molecular level. It is applausive that, aided by VS, we
obtained these positive results only within 9 months. Taking the most active
compounds as leading scaffolds, the focus libraries syntheses by combinatorial
chemistry and crystal structures of the complexes determination by X-ray
crystallography are being performed.
Nevertheless, VS
are still facing many challenges. Most of the current docking methods such as
DOCK and AutoDock have not considered the flexibility of targets (mostly
proteins); there is no universal scoring function for ranking the binding
affinity between small molecules and targets. Solving these two problems is the
main task in developing new virtual screening methods based on docking.
Combinatorial chemistry will produce more than 10200 compounds in the coming
years. VS can’t finish the screening of such a large number of compounds in a
short period even use the fastest supercomputer in the world. Therefore,
integrating docking approaches with molecular diversity and druglikeness
analyses is a tendency of VS development.
( Division of
Molecular Microbiology, Center of Molecular Biosciences, University of the
Ryukyus, Nishihara, Okinawa 903-0213, Japan, *e-mail: [email protected])
Transgenic plant technology is particularly useful to
increase food production levels by introduction of new traits, such as
resistance to insecticide, insect pests, high salinity, cold temperature and
drought. These what we call “first-generation” transgenic plants increase food
productivity and may eventually contribute to solve food crisis worldwide.
However, within the last decade scientists focused more on the development of
transgenic plants such as potatoes and rice, which are capable of synthesizing
recombinant proteins with pharmaceutical values. Examples include nutritional
proteins such as human milk proteins, monoclonal antibodies against bacteria
causing dental caries, and vaccine antigens like hepatitis B virus surface
antigen and cholera toxin. In contrast to the first-generation transgenic
plants, these “second-generation” transgenic plants may contribute to
large-scale and cost-effective production of medically valuable proteins.
Recently we have also demonstrated potential
usefulness of yeast for oral delivery systems to induce local as well as
systemic humoral immune responses against expressed antigens. Yeast generally
recognized as safe for food industry could be used for foreign gene expression
as well as oral delivery systems for vaccine antigens. In both plant and yeast
gene expression systems, we are particularly interested in using cholera toxin
B subunit (CTB) that is known to function as an effective mucosal antigen as
well as a carrier for genetically conjugated antigens. Orally delivered CTB or
CTB fusion proteins expressed in plants and yeast could bind to GM1-ganglioside
receptor located on cells in the gut-associated lymphoid tissues to induce
specific local and systemic immune responses. We demonstrated that oral
administration of these genetically modified plants and yeasts could
potentially be used for oral vaccines against infectious diseases like Japanese
encephalitis and malaria as well as autoimmune diseases such as
insulin-dependent diabetes mellitus. In addition, such technology could be
applied not only for human use but also for veterinary medicine.
Identification and experimental verification of novel genes in human fetal hippocampus
Jing-Jing Cai1&, Xiao-Jing Sun1,2*, Ke-Yue Ding1,2,
Yan Zhang1,2, Da-Long Ma1,3#,
Yan Shen1,2
( 1China
National Center of Human Genome Research, #3-707, Yong Chang North Road, BDA,
Beijing 100176, China;
2Institute of Basic Medical Sciences, the
Chinese Academy of Medical Sciences, Beijing 100005, China;
3Peking University Center for Human Disease
Genomics, 38 Xueyuan Road, Haidian District, Beijing 100083, China;
&e-mail: [email protected],*e-mail:
[email protected], #e-mail: [email protected] )
The abundant human genomic sequence data and
fragmental ESTs database are mostly unexplored resources for biomedical
investigation and characterization. The big resources provide an opportunity to
accelerate our understanding of ourselves, human beings. However, optimal
utilization of this information requires the development of methods for
harvesting novel genes, verifying by RT-PCR and evaluating protein function. We
are interested in human brain, especially human fetal hippocampus, because
hippocampus plays a central role in memory processes and these processes may be
related to development from fetal hippocampus to adult hippocampus.
An ESTs database was generated based on sequence data
of human fetal hippocampus cDNA library, which was constructed in our previous
study. It showed that 49% ESTs in this database are unknown, while only 27% are
known genes. With almost 10 000 raw ESTs analyzed, we selected 20 confirmed
seed ESTs, which might be the fragment of the novel protein-coding genes, for
further extending with overlapping human ESTs to find full-length cDNA in
silico. Some of these potential human genes were analyzed by BLASTN
searches in GenBank nr database and human ESTs database, then localized on the
chromosome uniquely and confirmed as the novel genes. These identified novel
human cDNA sequences covered 1000 – 4000 bp in full-length, with complete open
reading frame encoding small protein of 200 – 500 amino acid residues. All the
genes have certain functional domains. Based on predicted functional clues, 6
candidate genes had been amplified in Human Fetal MTC panel, and almost all
expressed in human fetal brain cDNA library. The subcellular localization and
some functional screening are in process.
Taking advantage of cloning full-length cDNA in
silico and finding functional clues by computer prediction, more novel
genes would be explored in the human genome, and our experiment proof verified
the effectiveness of this protocol.
Xiao-Jing Sun1,2*, Jing-Jing
Cai2, Ke-Yue Ding1,2, Pei-Guo Ding3, Da-Long Ma2,3#, Yan Shen1,2
(1 National
Laboratory of Medical Molecular Biology, Institute of Basic Medical Science,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
100005, China;
2 Chinese National Human Genome Center,
Beijing 100176, China;
3 Laboratory of Medical Immunology,
School of Basic Medical Science, Peking University, Beijing 100083, China;
*e-mail: [email protected], #e-mail:
[email protected])
With the progress of the human genome project, one of
the key results of the monumental work is that there might be fewer than 30 000
protein-coding genes in the human genome . However, it is not yet routine to
identify all possible mammalian genomic regions that are transcribed. Three
basic approaches are combined for gene prediction: direct evidence of
transcription by ESTs or mRNAs; indirect evidence of based on sequence
similarity to previously identified genes and proteins; and ab initio
recognition of groups of exons on the basis of hidden Markov model. The special
powerful material, dubbed expressed sequence tags (ESTs), has been used to
catalog the gene expression profile, and to identify novel genes. ESTs are
nucleotide sequences generated from single-pass cDNA sequences of the ends of
randomly selected clones from different tissue or cell type sources . Although
still fragmental, this large database now is an unexplored resource for
biomedical investigations involving gene identification and characterization .
The concept of comparative gene identification (CGI) has been used to search
human orthologous genes that have been identified in model organisms.
We have developed an efficient method in silico
cloning process for harvesting novel human genes. We used ESTScan program to
detect, evaluate and reconstruct coding regions in the ESTs sequence data. The
corresponding amino acid sequences were then searched against the GenBank
nr-protein database; all the similarity hits were parsed in a homology analysis
workbench. Some “seed” ESTs might be the fragment of the novel protein-coding
genes. These “seed” ESTs were mapped to human genome uniquely. Indeed, further analysis
of these ESTs in full-length cDNA cloning in silico, the mapping
information in UCSC, and the experiment proof, verified the effectiveness of
the strategy. Taking advantage of rare ESTs resources, and the effective
strategy, more novel genes would be explored in the human genome. To verify the
genes existence and function, we outline a new PCR strategy to clone the genes
from human multi tissue panel. By this method, we have obtained 70 sequences
already, and got 36 in success, 7 in 36 have variant transcripts. Now, we have
done a filtration in various cell lines and obtain some preliminary result.
Further researches are in process.
Metal-induced reversible structural interconversion of human mitochondrial NAD(P)+-dependent aalic enzyme
( Department
of Life Science, Yang-Ming University, Taipei 112, China, *e-mail:
[email protected] )
Human mitochondrial NAD(P)+-dependent
malic enzyme was strongly inhibited by Lu3+. The X-ray crystal
structures indicated a structural change between the metal-free and Lu3+-containing
enzymes. With the authentic crystal structures in hand, we characterized the
reversible slow binding mechanism and the structural interconversion between Mn2+-
and Lu3+-containing human mitochondrial malic enzymes. When Lu3+
was added, the activity of the human enzyme showed a downward curve over time,
similar to that of the pigeon enzyme. The rate of the transformation (kobs)
from the initial rate to the steady-state rate increased hyperbolically with
the concentration of Lu3+, suggesting the involvement of an
isomerization step. Lu3+ had a much higher affinity for the
isomerized form (Ki,Lu = 4.84 μmol/L) than that of the native
form (Ki,Lu = 148.4 μmol/L). When an excess of Mn2+
was added to the Lu3+-inhibited enzyme, assays of the kinetic
activity showed an upward trend, indicating reactivation. This result also
indicated that the reactivation was a slow process. Fluorescence quenching
experiments confirmed that the Lu3+-induced isomerization was
completely reversible. The dynamic quenching constants for the metal-free, Mn2+-containing,
and Lu3+-containing enzyme were 3.08, 3.07, and 3.8 (mol/L)-1,
respectively. When the Lu3+-containing enzyme was treated with
excess Mn2+, the dynamic quenching constant returned to the original
value [3.09 (mol/L)-1]. We proposed a model to interpret the
structural interconversion of malic enzyme. Binding of Mn2+ did not
induce any conformational change in the enzyme. The open form transformed to
the closed form only after substrate binding. Lu3+, on the other
hand, transformed the open form into a catalytically inactive form. Excess Mn2+
could replace Lu3+ in the metal binding site and convert the
inactive form back into the open form. This reversible process was slow in both
directions because of the same but opposite structural change involved.
Expression of foreign peptides with tobacco mosaic virus-based vector in plant
Lu-Bin Jiang,
Li-Gang Wu, Zhi-Ai Zhou, Mang-Mang Li, Hui-Hui Zhu, Zheng-Kai Xu*
( Shanghai
Institute of Plant Physiology & Ecology, SIBS, the Chinese Academy of
Sciences, Shanghai 200032, China, *e-mail: [email protected] )
Tobacco mosaic virus (TMV) is a very stable, rod
shaped, plus-sense, positive-stranded RNA virus, which functions very
efficiently in genome replication and gene expressions during the infection of
host plants. The amount of the expressed TMV coat protein (CP) could be
surprisingly accumulated up to 20 percent of the total protein in the host
cell. Based upon the crystallographic studies, the C-terminal portion of each
CP subunit in the viral particles protrudes outward from the TMV particles.
This may explain why some in frame insertions of various foreign short
peptide sequences to the C-terminal portion of the CP have no serious effects
on the virus particles assembly or the viral infectivity. The foreign short
peptides are suggested to stick out around the virus particle surfaces. This
feature may allow us to express some important short peptides such as epitope
peptides on the surfaces of the TMV particles, which may be utilized as
vaccines to control certain important diseases.
In our laboratory, we have initiated the vaccine studies on a very important animal virus, foot-and-mouth disease virus (FMDV) that causes very serious diseases in many important meat and milk producing domestic animals. The sequences of the foreign peptides with different length, which specify the dominant immunogenic site (amino acid residues 141 – 160) located in the FMDV structural protein VP1, were inserted into the region near the C-terminal of TMV CP gene. Those fused CPs can be expressed with high efficiency similar to wild-type CP in the tobacco systematically infected by above-mentioned recombinant TMV. Protection assay of guinea pig showed that the recombinant TMV, TMV-11 and TMV-14, whose fused CP were produced by inserting nucleotide sequences encoding 11 and 14 FMDV epitope peptides respectively between amino acid residues Ser154 and Gly155 of wild type TMV CP had the strong immunogenicity specific to FMDV. And the protection index value of the antisera raised from the guinea pigs that were injected with TMV-11 and TMV-14 was determine to be at least 2.0 on sucking mice. However, it was found that the level of the expression of the recombinant CP (TMV-20 CP) fused with the FMDV epitope peptide of twenty amino acids was greatly decreased to 1/5 of that of wild-type CP, and the corresponding recombinant TMV particles were apparently unstable. Computer analysis showed that too long foreign peptide fused to CP would produce the great effects to the second structure of the recombinant CP, which may interfere with the assembly of recombinant virus particles and then exert its infection efficiency. It is possible that the greater effect will be produced along with the longer foreign sequence fused to CP. According with this result, any published reports did not show that there were some TMV vectors carrying foreign peptides of more than 20 amino acids can express the corresponding fused CPs in host plants efficiently.
As to study the possible relationships belong to TMV
genomic sequence, the length of the fused foreign sequence and the assembly of
recombinant TMV particles, the particles of the recombinant virus TMV-20 were
mutagenized with sodium nitrite and then inoculated the tobacco. We found that
the 4 C-terminal amino acids of TMV CP might not be crucial for efficient
replication and assembly of recombinant TMV particles in host plant. In
addition, we also found the two new sites for fusion of foreign peptides near
the carboxyl terminal of TMV CP. Furthermore, the amino acid residues Trp152
and Thr153 maybe do not play important role in efficient replication
and assembly of recombinant TMV particles too. On the basis of our works, we
reported several recombinant TMV vectors which can express 11 to 28 amino acids
FMDV epitope peptides with high efficiency similar to that of w.t. TMV by inserted
the peptides into the region near the C-terminus of TMV CP in tobacco. The
recombinant TMV can be obtained without modifications during purification with
a yield of about 0.5 – 1 g virus from 100 g systematically infected fresh
leaves. And the recombinant virus appeared very stable during store at 4 ℃,
the short FMDV peptides still retained in the recombinant coat protein proved
by SDS-PAGE and western blot analysis. Currently the fused CPs isolated from
the purified recombinant TMV are having been utilized to test the effects of
protecting animal (pig) from FMDV infection.
Yu sun,
Xiao-Shu Gao, Qing-Qi Zhang, Zheng-Kai Xu*
（ Shanghai Institute of Plant
Physiology & Ecology, SIBS, the Chinese Academy of Sciences, Shanghai
200032, China, *e-mail: [email protected] ）
Dunaliella salina is a unicellular green alga without
cell wall and one of the most halotolerant eukaryotes now known as it can live
in the environments with salinity ranging from 50 mmol/L to 5 mol/L NaCl. The
extraordinary feature makes it an intriguing system for elucidating the
mechanism involved in its halotolerance. However, lacking a reliable
transformation system is the main obstacle in the molecular study of this
organism. Based on the transformation studies on other green algae, we
developed a strategy for the transformation of D. salina. The strategy
involved the following steps: (1) Cells of D. salina were treated with a
chemical mutagen and then cultured on a chlorate-containing medium to enrich
chlorate tolerant mutants. By further careful screening, about seventy
individual mutants were found deficient in nitrate assimilation. Further NR
activity assay were performed on twelve mutants, none of them exhibited NR
activity even after nitrate induction. For 2 years of observation, 45
individual mutants still maintained the NR- phenotype in their progenies. (2)
An endogenous NR cDNA and a genomic fragment (16 kb) containing the NR gene
were isolated from the D. salina cDNA and genomic libraries. The cDNA
and the genomic fragment were then individually cloned into a ble
gene-containing vector to form pUCNRT124S and pDSNR which were used for the
transformation of the NR- mutants. (3) The electroporation parameters were
optimized according to the cell viability and the quantity of foreign DNA
maintained in the cells. Using ble gene as a selectable marker, forty
independent isolates of D. salina were obtained with apparent
Zeocin-resistance. However, after longer incubation, only 10% of the progeny
cells from one isolate retained the resistance. Southern analysis suggested
that the transformed DNA was apparently associated with the portion of
exochorosmomal DNA.
Another approach has been in progress for the
transformation of D. salina in our laboratory. With this approach,
wild-type D. salina cells were transformed with a vector pUCNR5KL25 in
which the genomic fragment containing the NR gene from D. salina
was inserted to flank the ble gene cassette. With this vector, the
resulting transformants would be resistant to Zeocin. It is also possible that
some transformants would be Zeocin resistance and NR defective, with a very low
frequency, if the homologous recombination events occur in the region of the NR
gene. In one transformation experiment, one isolate has been obtained that
exhibits both of the NR– and Zeocin resistance phenotypes.
Isolation and characterization of salt tolerance related genes from Dunaliella salina
Xiao-Shu Gao,
Qi-Yun Li, Zhi-Yong Yang, Yu Sun, Yan-Tao Liu, Hong-Yun Dong, Qing-Qi Zhang, Zheng-Kai Xu*
( Shanghai
Institute of Plant Physiology & Ecology, SIBS, the Chinese Academy of
Sciences, Shanghai 200032, China, *e-mail: [email protected] )
Dunaliella salina (Volvocales, Chlorophyta) is a
unique unicellular green alga with extreme halotolerance, it can even live in
the environment saturated with NaCl (5 mol/L). It is therefore an ideal
experimental system to investigate the mechanism underlined the halotolerance.
To identify genes related to the halotolerance, two
specific cDNA libraries were constructed using SSH (suppression subtractive
hybridization) technique in our lab. The two SSH libraries each represents the
cDNA fragments expressed in Dunaliella salina cultured at constant 0.5 mol/L
NaCl or cultured for 24 h after an osmotic shock from 0.5 mol/L to 2.0 mol/L
NaCl. With the SSH cDNA fragments as probes, we have isolated several
full-length cDNA clones which probably encode two sodium-dependent phosphate
transporters, two carbonic anhydrases, one trehalose phosphate
synthase/trehalose phosphate phosphatase, one glucose-6-phosphate isomerase,
and three non-coding RNA sequences, respectively.
The two sodium-dependent phosphate transporter cDNAs
(DsNa+/Pi T1, DsNa+/Pi T2) encode a 672 and 675 amino
acids protein with a putative topology of 9 transmembrane domains interrupted
by a large hydrophilic loop between TM6 and TM7 respectively. A highly
conserved sequence (GANDVAN) in all the sodium-dependent phosphate transporters
found in eubacteria, archea, fungi, plants, and animals was also existed in
both of the N- and C-domains. A peptide (APAPTNGGGGC), probably a lipid anchor
for membrane fixation, was also present in DsNa+/Pi T1. The high
homology to PHO89 (NP-009855, a Na+/Pi transporter gene in yeast)
suggested both DsNa+/Pi T1 and DsNa+/Pi T2 were the
putative Na+/Pi transporter genes in D. salina. Northern analysis
revealed that the expressions of the two genes were induced by hyper-osmotic
shock from 1.0 mol/L to 2.0 mol/L NaCl and the transcript was accumulated to
the maximum 3 h after the osmotic shock.
Four genomic fragments were identified by southern analysis that possibly belonged to a carbonic anhydrase gene family in Dunaliella. Two genes DsCA1 and DsCA2 from this family have been cloned and sequenced. DsCA1 encodes a carbonic anhydrase that consists of two functional domains with high homology to that reported by Fisher et al. (1998). The expression of this gene is induced by an osmotic shock from 0.75 mol/L to 3.5 mol/L of NaCl, the transcript reached to the maximum 24 h after the osmotic shock. Different from DsCA1, DsCA2 encodes a putative carbonic anhydrase that has only one functional domain but with an unusual domain at the C-terminus, the expression of this gene is apparently induced by the osmotic shock.
Mutations in a new scaffold protein Sans cause
deafness in Jackson shaker mice：Positional cloning and application
to human deafness
Yoshiaki
Kikkawa1, Hiroshi Shitara1, Shigeharu Wakana2,
Yuki Kohara1, Toyoyuki Takada1, Mieko Okamoto1,
Choji Taya1, Kazusaku Kamiya3, Yasuhiro Yoshikawa3,
Hisashi Tokano4, Ken Kitamura4, Kunihiko Shimizu5,
Yuichi Wakabayashi6, Toshihiko Shiroishi2,5, Ryo Kominami6,
Hiromichi Yonekawa1*
(1 Department
of Laboratory Animal Science, The Tokyo Metropolitan Institute of Medical
Science (Rinshoken), Tokyo 113-8613, Japan;
2 Mouse Functional Genomics Research
Group, RIKEN Genome Science Center, The Institute of Physical and Chemical
Research, Yokohama 244-0804, Japan;
3 Department of Biomedical Science, The
University of Tokyo, Tokyo 113-8657, Japan;
4 Department of Otolaryngology, Tokyo
Medical and Dental University, Tokyo 113-8519, Japan;
5 Mammalian Genetics Laboratory,
National Institute of Genetics, Mishima 411-8540, Japan;
6 Department of Gene Regulation Division
of Molecular Biology, Niigata Graduate School of Medicine & Dental Science,
Niigata 951-8510, Japan,
*e-mail: [email protected] )
Over 70 mouse mutations affecting the inner ear have
been mapped in the mouse genome. Recently, a number of mouse mutations have
been identified by positional cloning, which is providing a rich source of
models for human deafness. Moreover, these hereditary hearing-loss genes
identified through human and mice are useful for mechanisms underlying both
normal and pathologic processes and for understanding the normal process of
auditory development and sensory transduction. One of such mutant mice is
Jackson shaker (js). The js mouse carries a recessive mutation causing
phenotypes such as deafness, abnormal behavior (circling and/or head-tossing)
and degeneration of inner ear neuroepithelia. Two alleles have been identified so
far, the original js and jsseal. A contig of three BAC clones was isolated by
positional cloning. Two of the clones rescue the js phenotype by BAC
transgenesis. Analysis of transcripts in an overlapping region of the two
clones revealed a gene encoding a new scaffold-like protein, Sans, that showed
mutations in the two js mutants. One was a guanine nucleotide insertion in the
original js allele and the other a 7-base insertion in the jsseal allele. Both
insertions are predicted to inactivate the Sans protein by frameshift mutations
resulting in a truncated protein lacking the C-terminal SAM domain. Sans was
highly expressed in inner and outer hair cells of cochlea, where abnormalities
are found in js mice. The existence of major motifs, ankyrin repeats, and a SAM
domain, suggests that the Sans protein mediates protein-protein interaction
with cytoskeletal structures that may be involved in signal transduction in the
cochlear hair cells. Similar mutations in the human ortholog were found in the
affected members in the Usher syndrome type IG. families, suggesting that the
deafness genes including our Sans will be a powerful tool for post-natal
diagnosis for human deaf patients.
Two or three IPG strips running on one SDS gel method
to improve the reproducibility, resolution power and high-throughput of
two-dimensional electrophoresis
Quan Yuan, Jie
An, Ding-Gan Liu, Fu-Kun Zhao*
( Key
Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai
200031, China, *e-mail: [email protected])
Two-dimensional
electrophoresis (2-DE) is used to study changes in cellular protein expression,
for detection of disease-related proteins, and in a great number of other
implications, since it is the only method currently available that is capable
of simultaneously separating thousands of proteins for quantitative comparison.
In this paper, we report a new IPG strip application, called “two or three IPG
strips running on one SDS gel” (TISROG) method. Through comparing the 2-DE
patterns of the same sample, the different state samples and the same sample in
the different second dimensional SDS running systems (Ettan Dalt twelve and SE
600) by TISROG method, we found this new method can not only improve the
reproducibility and resolution power of 2-DE pattern, but also achieve
high-throughput which is helpful to automatic proteomic research.
Proteomics analysis of differentially expressed proteins in the neural differentiation of P19 cells induced by all-trans retinoic acid
Jie An#, Quan Yuan, Ke Tang, Li
Liu, Chen Wang, Nai He-Jing, Fu-Kun
Zhao*
( Key
Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai
200031, China, #e-mail: [email protected], *e-mail: [email protected])
The P19 embryonal carcinoma (EC) cell line is a
widely used model system for analysis of neural development and differentiation
process that are difficult to study in mammalian embryos. They are pluripotent
stem cells and can be induced to differentiate in vitro into multiple
cells, including neurons. In this study, we have analyzed the protein
alterations during the differentiation stage of P19 cells into neurons induced
by all-trans retinoic acid (ATRA) in vitro by two dimensional (2-D) gel
electrophoresis and mass spectrometry. Image analysis of silver stained 2-D
gels revealed that 10 proteins showed significant expression change during P19
cells differentiation. Mass spectrometry is utilized to identify these protein
spots. The result indicated that these proteins maybe related to the neural
differentiation of the P19 cell.