Characterization of zebrafish vitellogenin gene family for potential development of receptor mediated gene transfer method 2

Chapter 2. Zebrafish vtg family

Chapter 2.
Characterization of the zebrafish vtg
family: sequencing, mapping and
phylogenetic analysis

27


Chapter 2. Zebrafish vtg family
Abstract

Vitellogenins (Vtgs) are precursors of yolk proteins in oviparous species and are cleaved
into three portions upon uptake by oocytes, lipovitellin I (LVI), phosvitin (PV) and
liopovitellin II (LVII). In the present study, we found that the zebrafish genome contains
at least seven vtg genes (vtg1-7) encoding heterogeneous vitellogenins with three distinct
groups: group A Vtgs (Vtg1, 4-7) contain all three major portions but lack the C-terminal
half of LVII; group B Vtg (Vtg2) is the only one including intact three portions; group C
Vtg (Vtg3) lacks both PV and the LVII C-terminal half. The seven vtgs were located in
two different chromosomes: one (vtg3) in LG11 and the rest closely linked in LG22.
Phylogenetic analysis indicated that the expansion of group A vtgs in zebrafish is lineage
specific, whereas gene duplication forming precursors of three groups of vtg may occur
before the radiation of teleost fish and may through whole chromosome or genome
duplication.

28


Chapter 2. Zebrafish vtg family
2.1. Introduction

In oviparous vertebrates, yolk is critical for embryonic development as it is a rich source
of nutrients, including amino acids, phosphate, carbohydrates, lipids and vitamins. In
addition, maternally derived hormones such as thyroxine and T3 have also been detected
in embryonic yolk (Kobuke et al., 1987). Early studies on the composition of yolk
revealed two classes of egg yolk proteins: the phosphoserine rich glycosylated phosvitin
(PV) and lipid-binding lipovitellin (LV) (Wallace and Jared, 1968; 1969). Both types of
proteins are derived from a common precursor protein termed as vitellogenin (Vtg), which
was first coined by Pan et al. (1969). Vtgs are calcium and zinc-binding
phospholipoglycoproteins synthesized in hepatic parenchymal cells under the influence of
female sex steroid hormone, estrogen (E2) (Wallace and Jared, 1969; Montorzi et al.,
1995).

Vtgs are ancient proteins belonging to a multiple member family that includes a variety of
lipoproteins (Bryne et al., 1989). Mammalian apolipoprotein B, the large subunit of
mammalian microsomal triglyceride transfer protein and insect apolipophorin II/I
precursor are suspected to share a common ancestor with Vtgs (Baker, 1988a&b; Babin et
al., 1999). Vtgs are encoded by multigene families in essentially all oviparous species
examined. For example, Wahli et al. (1979) first reported that there were four vtgs in
Xenopus laevis. In the nematode Caenorhabditis elegans, six vtgs have been identified,
which are located on different chromosomes (Spieth et al., 1991 and references within).
The number of vtgs reported in different fish varies with species. It is believed that a
teleost genome contains 2-20 copies of vtgs depending on species. The presence of several

29


Chapter 2. Zebrafish vtg family
distinct vtg EST clones in the zebrafish (Danio rerio) also indicated that multiple copies of
vtgs exist in this fish species (Gong et al., 1997).

The genes or cDNAs encoding Vtgs have been described in many vertebrate species
including chicken, Xenopus, and several fish species (Chen et al., 1997 and references
within). Vtgs are among the largest proteins and contain up to 2,139 amino acids with a
predicted molecular weight of 250 kDa before post-translational modification (Chen et al.,
1994a). After a Vtg precursor is internalized into the ovary by receptor-mediated
endocytosis (RME), it is cleaved into LVI, PV and LVII (Wahli, 1988). PV is a serine rich
domain containing one or more stretches of serine residues. In Xenopus, the PV can be
further cleaved into two smaller phosphoproteins, phosvettes I and II (Wahli, 1988).

In genomic organization, despite similar length of Vtg proteins, C. elegans vtgs have 4-5
exons (Spieth et al., 1991) whereas Xenopus and chicken vtgs have 35 exons (Nardelli, et
al., 1987). The size of the corresponding exons between Xenopus and chicken vtgs was
highly conserved except for exon 23, which generally codes for the PV moiety in
vertebrates. Preliminary studies on Vtgs in invertebrates suggested that these organisms do
not have the PV domain (Spieth et al., 1985; Trewitt et al., 1992). However, a
contradictory report from Chen et al. (1994a) stated that two polyserine rich regions were
discovered in mosquito (Aedes aegypti) Vtg. Whether Vtgs without phosvitin also exist in
vertebrates is unknown. Therefore, it is imperative to investigate vtgs from a wide
diversity of organisms for a better understanding of the evolutionary relationships among
vtgs.

30


Chapter 2. Zebrafish vtg family
The purpose of the present study was to characterize the zebrafish (Danio rerio) vtg
multigene family, including identification of individual vtg members, elucidation of the
primary structures of Vtg proteins, mapping of vtg genes and inferring the evolutionary
relationships among Vtgs of various fish species. Based on this characterization, an
appropriate Vtg candidate will be selected for preparation of a DNA carrier, which will be

used in the development of a novel gene transfer method, receptor-mediated gene transfer
(see Chapter 4).

31


Chapter 2. Zebrafish vtg family
2.2. Materials and Methods
2.2.1. vtg cDNA clones and DNA sequencing
All vtg cDNA clones were obtained from our previous EST clones isolated from an adult
cDNA library (Gong et al., 1997). Longest clones representing vtg1 to vtg7 were
sequenced completely from both ends by automatic sequencing. The deduced amino acid
sequences of Vtg1-7 were determined by DNAMAN V 4.15 (Lynnon BioSoft) and
putative signal peptide cleavage sites were determined by a computer program SignalP
V1.1, accessible at The deduced amino acid
sequences of Vtg1-7 were used in Fasta search ( to
determine the most homologous Vtgs in other species for phylogenetic analysis.

For manual sequencing, SK primer (5’-CGCTCTAGAACTAGGATC-3’) was used for
direct sequencing of 5’ ends of cDNA inserts using the T7 Sequencing Kit (Pharmacia)
and rapid denaturation and annealing were performed according to the manufacturer's
instructions.

For

automatic

sequencing,

primers

of

SK,

T7

(5’-

GTAATACGACTCACTATAGGGC-3’) and various gene specific primers (not shown)
were used for complete sequencing of whole inserts of representative vtg cDNA clones.
ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer
Applied Biosystems) was used to prepare sequencing reactions and the cycle sequencing
reactions were performed on a GeneAmp PCR system 9600 (Perkin Elmer). Briefly, each
20 µl of reaction was composed of 8 µl of Terminator Ready Reaction Mix, 3.2 pmol of
primer and 200-500 ng of plasmid DNA. The reaction was performed for 25 cycles with
the following parameters for each cycle: denaturing at 96 ºC for 10 sec, annealing at 50 ºC
for 5 sec and extension at 60 ºC for 4 min. The sequencing reaction products were then
32


Chapter 2. Zebrafish vtg family
loaded onto polyacrylamide gel, followed by gel electrophoresis and data process using an
automatic sequencer (ABI Prism 377, Perkin Elmer).

2.2.2. Sequence alignment and phylogenetic analysis
Putative Vtg amino acid sequences were aligned by a multiple sequence alignment
program Clustal W ( with default parameters. Well aligned
regions were chosen from each sequence and used in phylogenetic analysis by the
parsimony method using the phylogenetic program PAUP v3.1 (Swofford, 1993). The

input file was in NEXUS format following the PROTPARS example included in the
PAUP program. One-hundred bootstrap replicates were attempted using the heuristic type
of search. The mosquito (Aedes aegypti) Vtg (GenBank accession No. AAA18221) or C.
elegans Vtg1 (GenBank accession No. U37430) was used as an outgroup in construction
of the phylogenetic tree. Sequence alignment was also performed by DNAMAN V 4.15
(Lynnon Biosoft).

2.2.3. 5' RACE PCR and partial genomic region amplification of vtg3
Because of the relatively high sequence divergence of vtg3, full length vtg3 cDNA was
isolated for further characterization. Briefly, a Marathon cDNA Amplification Kit
(Clontech) was used to construct an adaptor-ligated double stranded cDNA library from
total liver RNA of female zebrafish based on the manufacturer’s instructions. One genespecific primer (primer 1: 5'-GGTAACTCAAGTGGCCAAGT-3', Figs. 2-3 and 2-12) was
designed for the 5' RACE-PCR using a diluted adaptor-ligated liver cDNA library as
templates. The 5’ missing cDNA sequence of vtg3 was obtained by performing PCR using
primer 1 and an adaptor primer AP1 supplied by the manufacturer. In 50 µl of PCR
33


Chapter 2. Zebrafish vtg family
reaction, there was 29.8 µl of dH2O, 5 µl of 10 x PCR buffer, 5 µl of 10 x dNTPs (2 mM
each), 3 µl of 25 mM MgCl2, 1 µl each of primer 1 and AP1 (10 µM each), 5 µl of 1/5
diluted adaptor-ligated liver cDNA library and 0.2 µl of Taq DNA polymerase (5 U/µl).
PCR was performed on a DNA Thermal Cycler 480 (Perkin Elmer) with the following
parameters: 94 oC for 2 min; 35 cycles of 94 oC for 30 sec, 60 oC for 1 min and 72 oC for 2
min; finally 72 oC for 8 min. A PCR fragment of about 2-kb was recovered from agarose
gel and ligated into pT7Blue vector (Novagen). Subsequent transformation was carried out
using competent cells DH5α and resulting colonies were screened by PCR. The plasmid
harboring the retrieved 5’ sequence of vtg3 was sequenced from the multiple cloning site
by automatic sequencing.

To determine whether the vtg3 genomic sequence comprises a PV region, zebrafish
genomic DNA was extracted from a single fish according to a modified protocol reported
by Xu et al. (1999), which was based on a standard protocol by Sambrook et al. (1989).
Two primers (primer 2: 5' -TGCACACTATCTTCACGAA-3' and primer 3: 5' GCTGATGTATGAGTCCTAT-3', Figs. 2-3 and 2-12) flanking the putative missing PV
region were designed and used in PCR amplification of partial genomic sequence of vtg3.
Briefly, 250 ng of zebrafish genomic DNA was used as templates in 50 µl of PCR
reaction, which contained the same reagents as described for the 5’ RACE PCR except for
the template. PCR was also performed on the DNA Thermal Cycler 480 with the
following parameters: 94 oC for 5 min; 30 cycles of 94 oC for 30 sec, 54 oC for 1 min and
72 oC for 2 min; finally 72 oC for 8 min. A fragment of ~ 3.0-kb was amplified from the
genomic DNA and cloned into pT7Blue vector (Novagen). The 3.0 kb insert was then
sequenced completely by automatic sequencing.
34


Chapter 2. Zebrafish vtg family
2.2.4. Genome mapping of zebrafish vtgs
To map the seven vtgs, mapping PCR was carried out using the T51 radiation hybrid
panel, which consists of 94 radiation hybrids of zebrafish-hamster hybrid cell lines
(Research Genetics) (Kwok et al., 1998; Geisler et al., 1999). For vtg1, vtg4, vtg5 and
vtg7, their forward and reverse mapping primers were designed based on nucleotide
sequences in the 3’end coding regions and 3’ UTRs, respectively (Figs. 2-1, 2-4, 2-5 and
2-7). For vtg2, mapping primers were designed based on the sequence located in the
unique 3’ end coding region of cDNA clone A183 (Fig. 2-2). vtg3 mapping primers were
targeted to an intron of the vtg3 gene (Fig. 2-14B).

Mapping PCR was performed using Taq PCR Core Kit (Qiagen). 5 µl of 25 ng DNA (of
radiation hybrids or controls) was used in each 20 µl of reaction which contained 6.5 µl of
dH2O, 2 µl of 10 x PCR buffer, 4 µl of 5 x Q-solution, 0.4 µl of dNTPs (10 mM each), 1
µl each of forward and reverse primers (20 µM each) and 0.1 µl of Taq DNA polymerase

(5 U/µl). The PCR machine was programmed with the following parameters: one cycle of
initial denaturation at 95 °C for 3 min, 45 cycles of amplification at 94 °C for 30 sec, 55 °C
for 15 sec and 72 °C for 1 min and a final extension at 72 °C for 8 min. PCR products were
resolved on a 1.5% agarose gel and images were recorded under UV illumination. Most of
the reactions were performed in duplicate and some in triplicate. Consistent results from at
least two separate reactions were scored according to SAMapper codes ("1" – specific
band amplified, "0" – no band amplified, "R" – undecided) and submitted for analysis via
e-mail to :8082/rh/.

35


Chapter 2. Zebrafish vtg family
2.2.5. Zebrafish databases used
Data on mapped vtg EST clones was retrieved from the zebrafish EST database at
(Washington University Zebrafish Genome Resources Project).
Searching

of

the

UniGene

database

( and the Ensembl zebrafish
genome database v19.3.2 ( was carried out for
determining vtg clusters and potential novel vtgs in the zebrafish genome. As a
comparison,

fugu(Fugu

rubripes)

genome

database

v2.0

( was also searched to determining homologous
vtgs in this new model fish.

36


Chapter 2. Zebrafish vtg family
2.3. Results
2.3.1. Presence of multiple vtg cDNAs in zebrafish
2.3.1.1. Identification of seven distinct vtg sequences from vtg EST clones
In our preliminary zebrafish EST project, among the 401 randomly selected cDNA clones
from an adult zebrafish cDNA library, 42 clones were identified to encode Vtgs (Gong et
al., 1997). Among them, twenty four (57.1%) were derived from the same gene, which
was named vtg1; six of them (14.3%) from a second gene named vtg2; one (2.5%) from a
third distinct gene, vtg3; two (5%) from vtg4; seven (16.7%) from vtg5; one each (2.5%)
from vtg6 and vtg7 (Table 2-1). Thus, the zebrafish has at least seven distinct and
functional vtgs. From the frequency of the seven vtgs in the cDNA library, it seems that
vtg1 is the most abundantly expressed vtg. vtg2 and vtg5 have similar medial expression
levels, whereas the other vtgs are expressed at relatively low levels.

The longest vtg inserts in each of the seven groups of vtg EST clones were sequenced
completely, including A248 (vtg1), A183 (vtg2), A376 (vtg3), A391 (vtg4), A227 (vtg5),
A220 (vtg6) and A349 (vtg7). The nucleotide and deduced amino acid sequences of the
seven vtg cDNAs are shown in Figs. 2-1 to 2-7. Among the seven vtg cDNA clones, A248
(vtg1) has the longest insert of 3645 bp, covering the majority of the coding sequence
(CDS). A vtg1 genomic clone has been isolated recently in our lab and a 5’ end 573-bp
vtg1 CDS was retrieved after sequencing (Shan, 2002; Tong et al., 2004). Thus, a
combined full-length vtg1 cDNA was obtained (4199 bp, excluding 5’ UTR and poly-A
tail) and it encodes a complete Vtg1 of 1360 amino acid residues (Fig. 2-1). The clone of
vtg3 (A376) is the second longest one with an insert of 2156 bp. After 5’ RACE-PCR,

37


Chapter 2. Zebrafish vtg family
Table 2-1. Percentage identity between vtg EST sequences and full-length cDNA
sequences of vtg1-7
A8**
A17
A22
A30
A67
A80
A87
A119
A126
A139
A183
A186

A192
A193
A220
A227
A248
A250
A252
A253
A256
A257
A259
A269
A272
A290
A295
A296
A300
A306
A342
A349
A368
A371
A376
A377
A381
A391
A397
A401
A419
A436

vtg1*
(A248)
92% (315)
47% (230)
96% (293)
95% (309)
93% (275)
98% (269)
97% (278)
72% (143)
98% (240)
100%(207)
86% (316)
96% (207)
97% (241)
92% (287)
91% (237)
91% (249)
100%(325)
99% (222)
97% (318)
96% (268)
100%(208)
100%(260)
99% (250)
92% (328)
97% (201)
98% (207)
91% (219)

81% (253)
97% (244)
94% (215)
99% (258)
96% (221)
98% (306)
88% (306)
53% (339)
98% (368)
100%(218)
85% (216)
98% (197)
48% (223)
97% (178)
46% (312)

vtg2
(A183)
47% (312)
96% (224)
41% (293)
47% (303)
81% (275)
89% (238)
44% (275)
78% (143)
66% (242)
44% (207)
100%(319)
76% (208)

43% (239)
47% (287)
86% (227)
47% (244)
43% (318)
78% (222)
42% (318)
80% (261)
44% (206)
44% (256)
78% (244)
46% (323)
79% (200)
42% (207)
79% (218)
99% (253)
45% (244)
44% (217)
43% (256)
81% (221)
88% (289)
98% (265)
43% (337)
44% (360)
48% (214)
72% (216)
47% (193)
98% (224)
88% (178)
96% (313)

vtg3
(A376)
46% (312)
45% (230)
43% (292)
40% (308)
41% (273)
42% (267)
43% (277)
40% (142)
45% (242)
43% (208)
51% (316)
52% (208)
48% (240)
44% (288)
43% (235)
47% (247)
40% (325)
49% (222)
39% (316)
48% (265)
40% (208)
47% (259)
43% (249)
46% (328)
52% (200)
43% (205)
43% (218)

46% (252)
43% (244)
48% (216)
41% (256)
53% (217)
48% (304)
47% (304)
100%(341)
44% (368)
48% (219)
49% (214)
48% (195)
47% (218)
40% (176)
47% (312)

vtg4
(A391)
45% (315)
52% (224)
44% (292)
48% (299)
45% (272)
44% (263)
43% (280)
95% (110)
59% (239)
43% (204)
40% (315)
94% (207)

45% (238)
40% (290)
45% (232)
49% (246)
44% (325)
96% (222)
43% (317)
97% (268)
46% (209)
46% (257)
95% (250)
43% (320)
94% (201)
50% (204)
95% (218)
81% (253)
48% (238)
48% (211)
44% (257)
94% (221)
43% (304)
49% (306)
43% (338)
43% (364)
40% (215)
100%(216)
43% (196)
49% (218)
45% (174)
43% (306)

vtg5
(A227)
93% (315) †
48% (227)
43% (292)
94% (309)
87% (275)
93% (269)
92% (278)
89% (140)
69% (237)
95% (207)
86% (313)
94% (207)
43% (240)
96% (287)
92% (237)
100% (249)
45% (317)
96% (222)
95% (318)
100%(268)
95% (208)
97% (260)
95% (250)
95% (328)
94% (201)
96% (207)
97% (218)

81% (253)
95% (244)
40% (216)
39% (256)
95% (221)
90% (304)
86% (307)
46% (337)
95% (368)
92% (218)
92% (215)
99% (197)
47% (219)
95% (178)
43% (309)

vtg6
(A220)
49% (316)
50% (229)
42% (295)
41% (305)
86% (275)
90% (247)
44% (279)
73% (140)
77% (240)
45% (204)
83% (319)
93% (205)

45% (240)
39% (283)
100%(237)
47% (249)
45% (324)
96% (219)
41% (317)
97% (265)
40% (210)
44% (258)
95% (250)
41% (322)
93% (198)
41% (208)
95% (215)
82% (250)
44% (242)
48% (215)
43% (252)
95% (218)
88% (299)
83% (274)
45% (336)
42% (368)
43% (217)
82% (215)
48% (195)
50% (223)
94% (178)
50% (314)

vtg7
(A349)
43% (314)
43% (230)
39% (294)
44% (301)
38% (272)
41% (269)
47% (274)
50% (144)
42% (241)
41% (205)
37% (317)
93% (207)
44% (240)
41% (285)
43% (233)
43% (247)
46% (316)
95% (209)
40% (315)
95% (234)
47% (208)
45% (295)
43% (249)
39% (329)
92% (201)
45% (205)
92% (218)

81% (253)
40% (243)
44% (211)
43% (252)
100%(221)
43% (306)
43% (305)
44% (341)
41% (364)
43% (213)
44% (211)
47% (193)
48% (223)
45% (178)
40% (307)

†Clones falling into one of the seven vtg categories are indicated by bold numbers (with ≥93%
sequence identities). Alignment range (bp) is listed in brackets. Note that <50% sequence
identities were observed for many clones, which was due to the lack of overlapping region.
*Full-length vtg cDNA sequence
**Partial vtg EST sequence

38


Chapter 2. Zebrafish vtg family

1
1
91

26

vtg1RTF1
CACAAACCACCAGCAATGAGAGCTGTTGTGCTTGCCCTGACGGTAGCCCTTGTGGCGAGTCAACAGTTCAACCTTGTTCCCGAGTTTGCC
M R A V V L A L T V A L V A S Q Q F N L V P E F A

181
56

CATGATAAGACCTATGTGTACAAGTATGAGGCTTTGCTCTTGGGTGGTCTTCCTCAAGAAGGTCTCGCCAGAGCAGGTATCAAAGTCAGC
H D K T Y V Y K Y E A L L L G G L P Q E G L A R A G I K V S
vtg1RTR1
AGCAAGGTTCGTCTGAGTGCCATGACAGAGAACACCTACCTGATGAAGCTTATGGATCCTCTACTCTACGAGTATGCTGGCACTTGGCCC
S K V R L S A M T E N T Y L M K L M D P L L Y E Y A G T W P

271
86

AAGGATTCATTTGTTCCTGCCACTAAGCTCACCTCAGCACTGGCTGCTCAGCTTCAGATCCCCATCCAGTTTGAGTATGCTAATGGTGTG
K D S F V P A T K L T S A L A A Q L Q I P I Q F E Y A N G V

361
116

GTTGGCAAGGTTTTTGCCCCAGCAGGAGTCTCTCCTACTGTCATGAACTTGCACAGAGGTATCCTCAACATCCTTCAGCTCAACCTCAAG
V G K V F A P A G V S P T V M N L H R G I L N I L Q L N L K

451
146

AAGACCCAGAACATCTACGAGCTGCAAGAGGCTGGAGCTCAGGGAGTGTGCAGGACACACTATGTCATCAATGAGGATCCAAAAACCAAC
K T Q N I Y E L Q E A G A Q G V C R T H Y V I N E D P K T N

541
176

CACATTATTGTCACCAAGTCTAAGGATCTGAGCCACTGCCAGGAGAGAATCATGAAGGATGTTGGCTTGGCATACACTGAGAGGTGTGCT
H I I V T K S K D L S H C Q E R I M K D V G L A Y T E R C A

631
206

GAATGCACAGAGAGGGTCAAGAGTCTGATTGAAACTGCAACTTATAACTACATCATGAAACCAGCTGACAATGGTGCACTGATCGCTGAG
E C T E R V K S L I E T A T Y N Y I M K P A D N G A L I A E

721
236

GCAACAGTTGAGGAAGTGTATCAGTTCTCACCCTTCAATGAGATTCATGGTGCTGCAATGATGGAAGCAAAACAAACCTTGGCTTTTGTT
A T V E E V Y Q F S P F N E I H G A A M M E A K Q T L A F V

811
266

GAGATTGAGAAGACCCCTGTCGTTCCAATCAAAGCTGATTACATGCCCCGTGGATCCCTGCAGTACGAGTTTGCAACTGAGATTCTTCAG
E I E K T P V V P I K A D Y M P R G S L Q Y E F A T E I L Q

901
296

ACCCCCATTCAACTCATGAAGATCAGTGATGCACCAGCCCAGATTGTTGAGGTCCTGAAGCACTTGGTTTCAAACAATAAAGACATGGTC
T P I Q L M K I S D A P A Q I V E V L K H L V S N N K D M V

991
326

CATGATGATGCTCCATTCAAGTTTGTTCAGCTTGTCCAGCTCTTGCGTGTTGCCTCCTTGGAGAAAATTGAGGCTATCTGGTCTCAGTTT
H D D A P F K F V Q L V Q L L R V A S L E K I E A I W S Q F

1081
356

AAGGACAAACCAGTTTACAGGCGCTGGCTTCTGGATGCTCTTCCTGCTGTTGGTACACCAGTCATTATAAAATTCATCAAGGAGAAGTTC
K D K P V Y R R W L L D A L P A V G T P V I I K F I K E K F

1171
386

CGGGCTGGTGAATTTACCACTCCCGAGTTCATTCAGACTCTTGTGATTGCTCTGCAAATGGTCACTGCTGATCCTGAAACCATTAAAATG
R A G E F T T P E F I Q T L V I A L Q M V T A D P E T I K M

1261
416

ACAGCTAGTTTGGCTACTCATGAGAAATTTGCCACAATCCCAGCTCTGCGTGAAGTTGTCATGCTTGGATATGGTTCCCTGATTGCCAAA
T A S L A T H E K F A T I P A L R E V V M L G Y G S L I A K

1351
446

TACTGTGTTGCAGTTCCCACTTGCCCTGCTGAGCTCCTCAGGCCCATCCACGAGATCGCCACAGAGGCCATTTCTAAGAATGACATTCCT
Y C V A V P T C P A E L L R P I H E I A T E A I S K N D I P

1441
476

GAAATCACTTTGGCTCTGAAAGTTATGGGCAATGCTGGTCACCCTTCAAGTCTTAAGCCAATCATGAAGCTCCTTCCTGGACTGAGGACT
E I T L A L K V M G N A G H P S S L K P I M K L L P G L R T

1531
506

GCAGCTAATGCTTTGCCCATTAGAGTCCAGGTTGATGCCATCTTGGCCCTGAGGAACATTGCTAAGAAAGAGCCCAAACTGGTTCAGCCT
A A N A L P I R V Q V D A I L A L R N I A K K E P K L V Q P

1621
536

GTTGCCCTGCAGCTTGTTTTGGACAGAGCTCTCCACCCAGAGGTGCGCATGGTTGCTTGTATTGTGCTGTTTGAGGCTGAGCCTTCAGTG
V A L Q L V L D R A L H P E V R M V A C I V L F E A E P S V

1711
566

GCACTTGTCTCTAGTCTTGCTGGAGCTTTAAGGATTGAGCCAAACATGCATGTTGCAAGCTTTGCCTATTCCCACATCAAGTCCTTGACC
A L V S S L A G A L R I E P N M H V A S F A Y S H I K S L T

1801
596

AGAATCACTGCTCCTGATATGGCATCTGTTGCTGGTGCAGCTAATGTTGCAATCAAGCTTATGAGCCGCAAACTGGACAGACTTAACTAC
R I T A P D M A S V A G A A N V A I K L M S R K L D R L N Y

1891
626

CGTTACAGCAGAGCTTTTCAGATGGACTATTATTATACTCCTCTTATGATTGGAGCTGCTGGTAGTGCCTATATGATCAATGATGCTGCC
R Y S R A F Q M D Y Y Y T P L M I G A A G S A Y M I N D A A

1981
656

ACCATCCTGCCCAGAGCTGTTGTAGCTAAAGCTCGTGCTTACCTGGCTGGAGCTGCTGCTGATGTTATTGAGTTTGGTGTGAGAACTGGA
T I L P R A V V A K A R A Y L A G A A A D V I E F G V R T G

39


Chapter 2. Zebrafish vtg family
2071
686

GGAATCCATGAAGCTCTCCTAAAATCTCCTGCTGCAGATGAAAGTGCTGACCGTATCACAAAGATTAAGCGTACACTGAGAGCACTCACA
G I H E A L L K S P A A D E S A D R I T K I K R T L R A L T

2161
716

AACTGGAAGGCTTTGCCAACCGATAAACCACTAGCATCAGCCTATCTCAAAGTATTTGGACAAGAAGTGGCTTATGTCAACTTTGACAAG
N W K A L P T D K P L A S A Y L K V F G Q E V A Y V N F D K

2251
746

ACCATCATTGAAGAAGCCATACCGATGGCTACTGGACCCAAACCACGTGCACTGCTGAAGGAGGCTCTTAAAGCTTTGCAGGAAGGAGTT
T I I E E A I P M A T G P K P R A L L K E A L K A L Q E G V

2341
776

GCCTTCCAGTATGCTAAACCTTTGCTTGCAGCTGAAGTGCGTCGTATCTTGCCAACTGCAGTTGGTGTGCCCATGGAGTTCAGTTGGTAC
A F Q Y A K P L L A A E V R R I L P T A V G V P M E F S W Y

2431
806

ACTGCTGCAGTTGCTGCTGCAAGTGTCAATGTTCAGGCCACCATTACACCTGCTCTCCCTGAGAAATTGGAGTCCATGACTTATGAGCAA
T A A V A A A S V N V Q A T I T P A L P E K L E S M T Y E Q

2521
836

CTAAAGAAGACTGATGTTCAGTTCCAAGCTGAAGCTAGACCAAGTGTTGCTCTCCAGACATTTGCTGTGATGGGAGTTAACACTGCCTTC
L K K T D V Q F Q A E A R P S V A L Q T F A V M G V N T A F

2611
866

ATCCAAGCTGCTGTTATGGCGAGAGGAAAGATCCGTACAATTGCCCCTGGAAAAGTGGCAGCAAGAGCAGACATTCTCAAGGGCAACTAC
I Q A A V M A R G K I R T I A P G K V A A R A D I L K G N Y

2701
896

AAGGTGGACGCTCTGCCTGTTGAACTTCCTGAACACATTGCTTCTGCAAGCTTTGAGACTTATGCCGTGGTCAGAAACATTGAAGATCAC
K V D A L P V E L P E H I A S A S F E T Y A V V R N I E D H

2791
926

AGTGCTGAAAGGTCTGTTCCCCTGGTACCTGAATTGTCTCTGCAAAACTCCCAGGCATCTTATGCTGGTGATTTGTCATCTGAGATGTCA
S A E R S V P L V P E L S L Q N S Q A S Y A G D L S S E M S

2881
956

TCTGTTGCTTCAGTAAGAGCTCCTGCTCCATTTGACAGAACCCTCTGTTATGCTGTCCCATACATTGAAATCAAGGGATGTGTTGAGGTG
S V A S V R A P A P F D R T L C Y A V P Y I E I K G C V E V

2971
986

CACTCTCACAATGCTGCTTTTATCAGAAATTCCACTCTTTTCTACATAATTGGACACCACTCAGTCCGTGCTGCAGTGGCAAGAGCTGAA
H S H N A A F I R N S T L F Y I I G H H S V R A A V A R A E

3061
1016

GGTCCTGCAGTTGAAAGACTGGAGTTTGAAGTTCAAGTTGGTCCTAGAGCTGCTGAGAGGCTTGTTAAGCAAATCAACATCATTGATGAT
G P A V E R L E F E V Q V G P R A A E R L V K Q I N I I D D

3151
1046

GATACTCCAGAAGGACAGGCTTTCTTGTTGAAACTGAGGGAAATCCTGGACACTGAAGCTAAAAATGCACCTGTTTCTTCTGAAAGCAGC
D T P E G Q A F L L K L R E I L D T E A K N A P V S S E S S

3241
1076

AGCAGTCGTAACAGTCGCAGCAGCAGCAGCCGCAGCACCAGCACTAGCACCAGCTCAAGCTCAAGCTCAAGTTCAAGTTCAAGCTCAAGC
S S R N S R S S S S R S T S T S T S S S S S S S S S S S S S

3331
1106

TCCTCTATGTCCAGCTCTCGTATGTCTAAGACTGCCACCATCATTGAGCCTTTCAGGAAATTCCACAAAGATCGGTACTTGGCACACCAT
S S M S S S R M S K T A T I I E P F R K F H K D R Y L A H H

3421
1136

AGCGCCACAAAGGATACTAGCAGTGGAAGTGCTGCAGCTAGCTTTGAACAAATGCAGAAACAGAATAGATTCCTTGGAAATGATATTCCA
S A T K D T S S G S A A A S F E Q M Q K Q N R F L G N D I P

3511
1166

CCTGTTTTTGCTATCATCGCCCGTGCTGTTAGAGCTGACCAGAAGCTTCTGGGCTACCAACTGGCTGCTTACTTTGACAAACCAACTGCA
P V F A I I A R A V R A D Q K L L G Y Q L A A Y F D K P T A

3601
1196

AGAGTGCAACTGATAGTTTCCTCCATTGCTGAAAACGACAACATGAAGATCTGTGCCGATGGTGCTCTGTTGAGCAAGCACAAAGTCACT
R V Q L I V S S I A E N D N M K I C A D G A L L S K H K V T

3691
1226

GGCAAGTTTTCTTGGGGTGCAGAGTGCAAACAGTATGCAGTCTTTGCTAAAGCTGAAGCTGGTGTCCTGGGTGAATTCCCTGCTGCACGT
G K F S W G A E C K Q Y A V F A K A E A G V L G E F P A A R

3781
1256

CTAGAAGTGGAATGGGAGAGACTGCCAATAATTGTCACCACCTATGCCAAAAAGCTGGGTAAACACATCCTTACAGCAGCTTACGACACA
L E V E W E R L P I I V T T Y A K K L G K H I L T A A Y D T
vtg1MF1
GGATTCAGGTTTGAACGAGCAACGAACAGTGAGAAAGAGATTGAACTGACTGCAGCCTTGCCATCTCAGAGGTCCTTGAATATCATTGCT
G F R F E R A T N S E K E I E L T A A L P S Q R S L N I I A
XmnI
AGGATTCCAGAGATCACAATGTCAAAAAGAGATATTTATCTTCCCGTCGCTGTTCCCATCAATCCAGACGGAACTTTTTCCATTGAGACC
R I P E I T M S K R D I Y L P V A V P I N P D G T F S I E T

3871
1286
3961
1316
4051

1346
4141
4231

TATGAGGACTTTCTCGCCTGGATCCAGAAATATATCAAGGAGGAATAAGAAATGAATTATATTCTAAATTTCCACAAGAAAATATGGCCT
Y E D F L A W I Q K Y I K E E *
ATGTTCATGTGGAATTTTGACATTTTAATTAGACCACTGCTGTACTGCAATTAACAATAAATTTCCTGCAAGTTAAAAAAAAAAAAAAAA
vtg1MR1
AAA

40


Chapter 2. Zebrafish vtg family

Fig. 2-1. Nucleotide and deduced amino acid sequences of full-length vtg1 cDNA. It was obtained
by combination of vtg1 cDNA clone, A248 and the 5’ end missing sequence provided by Mr. Tao
Shan. The start of vtg1 cDNA clone is indicated by an unfilled arrowhead. The stop codon is
represented by an asterisk. The polyadenylation signal, AATAAA and 3’ end XmnI site are
underlined. Primer sequences for vtg1RTF1/vtg1RTR1 (for real-time PCR) and
vtg1MF1/vtg1MR1 (for mapping PCR) were underlined. Putative signal peptide cleavage site and
two intron positions are marked by an arrow and black arrowheads, respectively.

41


Chapter 2. Zebrafish vtg family
1
1

CCGTGCTGCAGTGGCAAGAGCTGAAGGTCCTGCAGTTGAAAAACTGGAGTTTGAAGTCCAAGTTGGTCCTAGAGCTGCTGAGAGGCTTGC
R A A V A R A E G P A V E K L E F E V Q V G P R A A E R L A

91
31

TAAGCAAATCAACATCATTGATGATGAAACTCCAGAAGGAAAGGACTTCCTGTTGAAACTACGGGAAATCTTGGAGACTGAAAGCAAAAA
K Q I N I I D D E T P E G K D F L L K L R E I L E T E S K N

181
61

TGCAACTGTCTCTTCTGGAAGCAGCAGCAGCCGCAGCAGCAGTAGCAGCCGCAGCAGCAGCAGCAGTAGCAGCACCAGCAACAGTACCAG
A T V S S G S S S S R S S S S S R S S S S S S S T S N S T S

271
91

TAGCAGCAGCTCAAGCTCAAGCTCAAGCTCTTCCATGTCCAGCTCTCGTTGGTCTAAGACTCTCACTAAAATGGAAGCCTTCAGGAAATT
S S S S S S S S S S S M S S S R W S K T L T K M E A F R K F

361
121

CCACAAGGATCAGTATAAGACACATCATGGAGACTCAAAAAGCAGTAGAAGCACTGGATCTAGCCTTGAGCAAATCCAGAAACAGTCTAG
H K D Q Y K T H H G D S K S S R S T G S S L E Q I Q K Q S R

451
151

ATATCTTGGGAATACTGTTCCACCTGTTTTTGCTATCATCGCCCGTGCTGTAAGAGTTGACCGGAAGTTGTTGGGATACCAGTTTGTGGC
Y L G N T V P P V F A I I A R A V R V D R K L L G Y Q F V A

541
181

TTTCTTTGACAAGCCATCTTCAAGAGTGCAACTCATTGCTTCTTCCATTGCTGAGAATGACAACTTTAAGTTCTGTGCGGATGGTGTCCT
F F D K P S S R V Q L I A S S I A E N D N F K F C A D G V L

631
211

GCTGAGCAAGCACAAAGTCACTTCCAAGGTTACCTGGGGTGCAGAGTGTAAAGAATATGCAGTGACTACTAAAGCCGAGGCTGGACTCCT
L S K H K V T S K V T W G A E C K E Y A V T T K A E A G L L

721
241

TGGAGAATTCCCAGCTTTCCGTCTAGAGTGGGAATGGGAAAGGCTTCCAATTATTTTCACCACCTACGCCAAAAAGCTGTCTAAGCACAT
G E F P A F R L E W E W E R L P I I F T T Y A K K L S K H I

811
271

TCCTATGGCGGCTTTGCAAGCAGGATTTAATGTTGAGAGAGCTAAGAACAGCGAGAAAGAGTTAGAACTTACGGTGGCCTTGCCATCTAA
P M A A L Q A G F N V E R A K N S E K E L E L T V A L P S K

901
301

GAGAACACTGAATGTAATTGTTAGGGTTCCAGAGATGACAATGTCAAGGATGGATATTCCTCTCCCAGTTACTGTCCCTATTAATCCAGA
R T L N V I V R V P E M T M S R M D I P L P V T V P I N P D

991
331

TGGAACCTTTGATGTTCATTTTTATGAGGATATTTACTTCAGAGCCCAAAACTATATCTACGATTACACCACTGCTCAATGCAGCATGAT
G T F D V H F Y E D I Y F R A Q N Y I Y D Y T T A Q C S M M

1081
361

GCAGGATACAATCAGCACCTTCAACAACAAAACATACAAGAATGAAATGCCTATTTCCTGCTACCAAGTCTTAGCCCAGGATTGCACATC
Q D T I S T F N N K T Y K N E M P I S C Y Q V L A Q D C T S

1171
391

TGAGCTGAAATTTGTTGCTCTGTTGAAGAAGGACGAAGAGTCTGAAAAGACCCACCTGAATGTTAAACTTGTTGACATTGATATTGACCG
E L K F V A L L K K D E E S E K T H L N V K L V D I D I D R
vtg2MF4
GTATACTTTGGGCACTGATGCAAAAGTTAAAATTAATGGACTGGAAGTTCCCATCAGCAGCCTTCCTTATCAGCATCCCTCAGGCTCCAT
Y T L G T D A K V K I N G L E V P I S S L P Y Q H P S G S I
vtg2RTF3
CCAGATCAGAGAGAAGGCTGATGGTTTGTCACTTTATGCTCCTAGTCTTGGGCTTCACGAAGTCTACTTTGCCAATGGTGACTGGAAGAT
Q I R E K A D G L S L Y A P S L G L H E V Y F A N G D W K I

1261
421
1351

451
1441
481

1621
541

CCAAGTTGCAGACTGGATGAAGGGACAGACTTGTGGACTTTGTGGAAAGGCTGATGGAGAAATCAAACAGGAGTACACCACACCCAGTGG
Q V A D W M K G Q T C G L C G K A D G E I K Q E Y T T P S G
vtg2RTR3
ATACCTGACCGAGAGCTCAGTCAGCTTTGCACACTCATGGGTGCTGCCTGCTGAGAGTTGCCGTGATGCCAGCCAATGCCGCATGAAACT
Y L T E S S V S F A H S W V L P A E S C R D A S Q C R M K L
vtg2MR4
TGAATCTGTCAAGTTTGAGAAACAGGTGATTTTGAACGGACAGGAATCAAAATGCTATTCCGTTGAGCCTGTGCTGCGCTGTCTGCCAGG
E S V K F E K Q V I L N G Q E S K C Y S V E P V L R C L P G

1711
571

CTGTGCACCAGTAAGAACCACACCTGTGACAGTTGGATACCACTGTATGTCCACTGCCTCTAATCTCAACATGCTAGCTGGAATCTACGA
C A P V R T T P V T V G Y H C M S T A S N L N M L A G I Y E

1801
601

GAAGAGTGTAGATTTGAGAGAGACAACAGATGCTCACGTGGCCTGTCGCTGCACTGAGCAGTGTGCTTAAATCCACTTCTTGGCTCAGTA
K S V D L R E T T D A H V A C R C T E Q C A *
vtg2MR1
GAGTATAACAACATTCTCTGAATCTGCTGTTACTTGTTTAGAATAAATCTGAATAAGCAATCTTAAAAAAAAAAAAAAAAAAA

1531
511

1891

Fig. 2-2. Nucleotide and deduced amino acid sequences of vtg2 cDNA clone, A183. The stop codon is
represented by an asterisk and the polyadenylation signal, AATAAA, is underlined. Primers sequences for
vtg2MF4/vtg2MR4 (for mapping PCR), vtg2RTF3/vtg2RTR3 (for real-time PCR) and vtg2MR1 were
underlined. Intron positions are marked by arrowheads. CGLC motif in LV II region is boxed.

42


Chapter 2. Zebrafish vtg family

1
1

GGCGGCTGTGTCTCTGTCTCCTGGTAGCCCTGGCTGCCAGCGAGATGGCAAATTATGAGCCTTTTCTGAACTCGCAAAAAACATACGAGT
R L C L C L L V A L A A S E M A N Y E P F L N S Q K T Y E

91
30

ATAAATATGAAGGATTGGTACAAGTGGGACGGGAACTGCCACACCTGGTTGAATCAGCGCTGAAACTGAGGTGCACTTTTAAAATCATTG
Y K Y E G L V Q V G R E L P H L V E S A L K L R C T F K I I

181
60

GAGAGTCACCACACACCTTTGTCCTTCAGGTCTCAAATGTAGACTTTGAAGATTTTAATGGCATACCTGGGAAAAGTGTCTTCAGCCCTT
G E S P H T F V L Q V S N V D F E D F N G I P G K S V F S P

271
90

CCAAAAACATCACTAAGTATCTGTCTGCCGAGATCAGCCAGCCAATCATTTCTGAATATTCTAAAGGACAAATTACTGACATTCGCACAG
S K N I T K Y L S A E I S Q P I I S E Y S K G Q I T D I R T

361
120

CACCTGGAGTCTCAAACACAGTTGTGAATATTGTGAGGGGGATCCTTGGATTTTTACAAGTCACGGTCAAAACCACACAAAGTTTTTACG
A P G V S N T V V N I V R G I L G F L Q V T V K T T Q S F Y

451
150

AACTAATTGAGTTGGGAATTCATGGTTTGTGTCAGAGCAGTTACACTGTTGATGAAGACTCTAACCCAAAAGAGTTGATAGTAACACGAA
E L I E L G I H G L C Q S S Y T V D E D S N P K E L I V T R

541
180

TTGTTGATATCACCAACTGTCAACAGCCAGCATCTTTGTACAGAGGTATGGCTCTTGCACCTGAAGACAAACTTAGCAAACAGAGAGGCG
I V D I T N C Q Q P A S L Y R G M A L A P E D K L S K Q R G

631
210

AAAGCGTTGTTTCCACTGTGAAACACACCTACACAGTGAAGTCCACAGCAGACGGTGGTCAGATTACTAAAGCATTTGCTCAGGAGCGCC
E S V V S T V K H T Y T V K S T A D G G Q I T K A F A Q E R

721
240

AATATTTCTCTCCGTTCAATGTAAAGGGAGGAAACTTCCGAATGTTGGCATTGCGGGACATTGAACTTCTTAAAGTTTCAGACACAACTG
Q Y F S P F N V K G G N F R M L A L R D I E L L K V S D T T

811
270

ACAAAGTAGTGACTGGACAGGTGCAGAGCAGAGGCAACCTGATGTATAAGACAAATAAGGACCTCAAACCAATACCTGTTGTGATGCTTA
D K V V T G Q V Q S R G N L M Y K T N K D L K P I P V V M L

901
300

ACCTGAACGACCCAGTGCCCAAGATTTTAGATTTAATCAAGCGCCTCGCACAGGCTAATATATATCATGTGGACAGTGAAACCAGCACAG
N L N D P V P K I L D L I K R L A Q A N I Y H V D S E T S T

991
330

AAATTCTGGACCTAATACAGTTGATGCGGGTAACAACACTTGATAATCTGGAGCATTTATGGAAGCAGGTCTCAGGAAATGATGAGCACA
E I L D L I Q L M R V T T L D N L E H L W K Q V S G N D E H

1081
360

GGCGGTGGTTCCTGGACTTGGTTGTGGAGGTAACAGATGAAAGAATTCTCAAATTCCTTGAGGCCAGATACAAAGCAGGAGACATCACAG
R R W F L D L V V E V T D E R I L K F L E A R Y K A G D I T

1171
390

CGAATGAGGCAGGACAAGCACTGGTAGTGGCATTTAACCACTTGTCCGCTGAGCCTGTGTCGGTGGCATTAGCTCAGGAGTCCCTGACCA
A N E A G Q A L V V A F N H L S A E P V S V A L A Q E S L T

1261
420

TTCCTTTCAGTAAATCCCATCCTCTCCTGTGGAACACTGTAGTTTTGGCATATGGATCTCTTGTACACAGATACTGTGTATATACTGACC
I P F S K S H P L L W N T V V L A Y G S L V H R Y C V Y T D

1351
450

CCTGCCCTATCACTGTGGTACAGCCATTGCTGAATATGGCTGCAAGTAGCCTAAGTAAAAACTCTGAGGATGAAATGGTCCTTGCGCTGA
P C P I T V V Q P L L N M A A S S L S K N S E D E M V L A L

1441
480

AGTCCTTGGGAAACGCAGCTCATCTTTCCAGCATCAAGACTCTCCTCAAGTTCCTTCCTGGATACTCCAATGGAGCTGAAAAACTTTCCA
K S L G N A A H L S S I K T L L K F L P G Y S N G A E K L S

1531
510

CCAGAGTGCAGGGAGCTGCAGTCCAGGCATTTAGGCTGCTTGCAAGCAGAGCCCCCCACAGTGTACAGGATATTGTCTTAAACCTCTTCG
T R V Q G A A V Q A F R L L A S R A P H S V Q D I V L N L F

1621
540

TACAGAAACATTTACCGGCTGAAATCCGCATGCTGGCCTGCATAGTTCTTCTAGAGACCATGCCGTCCACAGCTCTGATTTCAGTAGTAA
V Q K H L P A E I R M L A C I V L L E T M P S T A L I S V V

1711
570
1801
600

GTGAGGTCCTTCTAGAAGAGGCTGATCTGCAGGTTGCCAGTTTCTCCTACTCTCTGCTCAAAGGCTTTGCCAAGTCCCGTACCCCTGATA
S E V L L E E A D L Q V A S F S Y S L L K G F A K S R T P D
primer 1
ATCAACATCTATCTATTGCCTGTAATATTGCCATGAAGATTCTAACCCGTAAACTTGGCCACTTGAGTTACCGCTACAGCAAGAACCTGC
N Q H L S I A C N I A M K I L T R K L G H L S Y R Y S K N L

1891
630

ACTTTGACTGGTTCCATGATGACTTTTTATTTGGGACATCTGCTGATGTTTATATGCTTCAGAATGAAAGTCCCATCCCCACAAAACTTA
H F D W F H D D F L F G T S A D V Y M L Q N E S P I P T K L

1981
660

TGCTAAAGGGAAAATTTCATTTCATTGGGAGAATACTGCAATTTCTTGAGTTTGGTATCCGTGCAGATGGACTCAAAGATCTGTTTGCTG

M L K G K F H F I G R I L Q F L E F G I R A D G L K D L F A

43


Chapter 2. Zebrafish vtg family
2071
690

GAAAAATCCCAGAACTCACAAAAGATTTAGGAATCAGCGATTTGGCTTCCATATTGAAAATTCTCTCTAACTGGCAGAGCCTACCAAAAG
G K I P E L T K D L G I S D L A S I L K I L S N W Q S L P K

2161
720

ACAAGCCTCTTTTGACAGCTTATGCAAGAGTTTTTGGACAGGAAGCCTTTTTAATGGATGTAAGCAGAGACTCAGTTCAAAGCATCATAA
D K P L L T A Y A R V F G Q E A F L M D V S R D S V Q S I I

2251
750

AGTCTTTCAGTCCTTCTGCAGGAAAGGAGAGTAAAGTTTGGGAAAGGATTCAAGATGTTCAGAAAGGGACTTCATGGCACTGGACTAAGC
K S F S P S A G K E S K V W E R I Q D V Q K G T S W H W T K

2341
780

CGCATCTTGTGTATGAGGCTCGGTTCATACAGCCAACATGCCTGGGTCTCCCAGTTGAAATCAGCAAATATTATTCCGTAGTAAATGCTG
P H L V Y E A R F I Q P T C L G L P V E I S K Y Y S V V N A

2431
810

TTACAATGCAAGCAAAAGCTGAAATAAATCCTCCTCCAAAGGAACATCTGGGTGAACTGCTAAGCTCAGACATTTCCATGCAGACAGATG
V T M Q A K A E I N P P P K E H L G E L L S S D I S M Q T D

2521
840

GCTTTATCGGCGTGACAAAGGATCATTTTCTCTTCCATGGAATCAACACTGATCTTTTTCAGTGTGGAACTGAGCTGAAAAGTAAGGTTT
G F I G V T K D H F L F H G I N T D L F Q C G T E L K S K V

2611
870

CAATGGGCCTGCCGTGGGCATTTGACCTGAAAATAAATCCGAAAGAGCAAAAGTATGAAATGAACTTGACTCCAAGCAAATCGGTCACCG
S M G L P W A F D L K I N P K E Q K Y E M N L T P S K S V T

2701
900

AATTATTTTCAGTCAGTTCTAATGTGTATGCTGTTTCGAGAAACATTGAAGACCCAACCTCATCTAAAATAACACCCATGATGCCTGAGA
E L F S V S S N V Y A V S R N I E D P T S S K I T P M M P E

2791
930

3061
1020

CAGGGGAGTCCTGGCAGGGTGTGCCTCTGAGAATGTTACCCCCTTTAAGGGATGAACAGTCCAAAAAATCTGGAATGAAATTCAGGCAAT
T G E S W Q G V P L R M L P P L R D E Q S K K S G M K F R Q
primer 2
GTGCTGAAGCCAAAATTTATGGAACTGCACTCTGCATTGAGGCAGAAGCCAAACGTGCACACTATCTTCACGAATATCCTCTGTATTACT
C A E A K I Y G T A L C I E A E A K R A H Y L H E Y P L Y Y
vtg3RTF1
TACTGGGTGATACTCACTTTTCATACAGTTTAGAACCAGCAAAGGATGCAAAACCCATTGAAAAAATTCAGATTCAGGTCTCTGCCAGCA
L L G D T H F S Y S L E P A K D A K P I E K I Q I Q V S A S
vtg3RTR1
GACAACATCCTTCAGTAATGAGTGGAATGGTGAATCTCAACCAAAGGGTATTTAAGGAGAAAAGAGATGAGAACACCTCCTGTGAGGAAC
R Q H P S V M S G M V N L N Q R V F K E K R D E N T S C E E

3151
1050

GCAAAACATCCAGTTCACTCCCAGTAACTCAGGATCTAGATGTCACTCCAGACCCGGTAGTCACAGTAAAAGCACTTAGTTTAAGTCCAC
R K T S S S L P V T Q D L D V T P D P V V T V K A L S L S P

3241
1080
3331
1110

AAGCAAAACCCCTTGGCTATGAAGGCGTAGCCTTTTATCTGCCAACTGCCCAAAAAGACGACATTGAAATGATCGTCTCTGAAGTTGGTG
Q A K P L G Y E G V A F Y L P T A Q K D D I E M I V S E V G
primer 3
AAGAAGCAAACTGGAAAATGTGTGCCAATGCACATTTTGATAGGACTCATACATCAGCAAAGGCTCATCTCAGATGGGGTGCAGAGTGTC
E E A N W K M C A N A H F D R T H T S A K A H L R W G A E C

3421

1140

AAACATATGATGTTTCTATGAGAGTGTCCGCAGCGTGCCAACCAGAGTCCAAACCATCTATAAGCACAAAGATTAACTGGGGGACTCTGC
Q T Y D V S M R V S A A C Q P E S K P S I S T K I N W G T L

3511
1170

CCTCAGTATTCACAACAGTTGGTCAAATAGTTCAAGAGTATGTACCTGGCGTGTCTTACATTATGGGTTTCTACCAGAAAAATGAGGAAA
P S V F T T V G Q I V Q E Y V P G V S Y I M G F Y Q K N E E

3601
1200

ACCCAGAACGGCAGGCATCTGTCACCGTTGTAGCATCCTCACCAGAGACCTTTGACCTGAAAGTGAAAATTCCAGAGCGAACTATCTACA
N P E R Q A S V T V V A S S P E T F D L K V K I P E R T I Y

3691
1230

AAAAGAAAATTCCCTCACCAATTGAACTTGTGGGGATCGAAGCTGCAAATCTCACCATGTCAACTTAACAGGCTGATGTCATAGAACATT
K K K I P S P I E L V G I E A A N L T M S T *

3781
3871

TCTGGAGGATTAAACCGACAATGTGAAATGAATGGATCATCAAATGTTGTGAATTGTTTGTACATTTCAGACATTTGATTAATTATTGAT
TTGGATACTAAATATGTGCTTAAAAGCAACATATGTTAAGTACTCTAACAAATAAAACATGCATGAAGAAAAAAAAAAAAAAAAAAAA

2881

960
2971
990

Fig. 2-3. Nucleotide and deduced amino acid sequences of vtg3 near full-length cDNA, which is
composed of the sequence of vtg3 cDNA clone A376 and the 5’-RACE PCR retrieved sequence. The
start of vtg3 cDNA clone A376 is marked by an unfilled arrowhead. The stop codon is represented by
an asterisk and the polyadenylation signal, AATAAA, is underlined. Primer sequences for primers 1, 2,
3 and vtg3RTF1/vtg3RTR1 (for real-time PCR) were underlined. Intron positions are marked by black
arrowheads.

44


Chapter 2. Zebrafish vtg family
1
1

TAGCAGCAGCAGCAGTCGCAACAGCCGCAGCACCAGTAGCAGCAGCACCAGCACTATCAG
S S S S S R N S R S T S S S S T S T I S

61
21

CAGCGGCAGCAGCAGCAGCAGCAGCAGCTCAAGCTCATCCATGTCCAGCTCTCGCATGTC
S G S S S S S S S S S S S M S S S R M S

121
41

TAAGACTGCCACCATAATGGAGCCTTTTAGGAAATTCCACAAAGATCGGTACTTGGCACC
K T A T I M E P F R K F H K D R Y L A P

181
61

CCATAGTGCCACAAAGGATACTAGCAGTGGAAGCGCTGCAGCTAGCTTTGAACAAATGCA
H S A T K D T S S G S A A A S F E Q M Q

241
81

GAAAAAGAATAGATTCCTTGGAAATGATATTCCACCTGTGTTTGCTATCATCGCCCGTGC
K K N R F L G N D I P P V F A I I A R A

301
101

TGTTAGAGCTGACCAGAAGCTTCTGGGCTACCAACTGGCTGCTTACTTTGACAAACCAAC
V R A D Q K L L G Y Q L A A Y F D K P T

361
121

TGCAAGAGTGCAACTCATAGCTTCCTCCATTGCAGAAAACGACAACATGAAGATCTGTGC
A R V Q L I A S S I A E N D N M K I C A

421
141

TGATGGTGCTCTGCTGAGCAAGCACAAAGTTACTGCCAAGTTTTCTTGGGGTGCGGAGTG
D G A L L S K H K V T A K F S W G A E C

481
161

CAAACAGTATGCAGTCTTTGCTAAAGCTGAAGCTGGTGTCCTGGGTGAATACCCTGCTGC
K Q Y A V F A K A E A G V L G E Y P A A

541
181

ACGTCTAGAAGTGGAATGGGAGAGACTGCCAAAAATTGCCTCCACCTATGCCAAAAAGGT
R L E V E W E R L P K I A S T Y A K K V

601
201

GTCTAAGTACATCCTTAATGCAGCTTATGACACAGGATTCAGGTTTGAAAGAGCAACGAA
S K Y I L N A A Y D T G F R F E R A T N

661
221

CAGCGAGAAAGAGATTGAACTGACTGCAGCCTTGCCATTTCAGAAGTCCTTGAATGTCAT
S E K E I E L T A A L P F Q K S L N V I
vtg4MF1
TGCTAGGATTCCAGAGATCACAATGTCAAGAAGGGATATTTACCTCCCCGTCACTGTTCC
A R I P E I T M S R R D I Y L P V T V P

721
241
781
261

901

CATCAATCCAGACGGTACTTTCACCATTGATAAGGATTTTCTCTCCTGGATTCCCAAATA
I N P D G T F T I D K D F L S W I P K Y
AseI
TATCAATGAGGATTGAAAAATTAATAACATTCTACATTTTCACAAGAAAATATGAGATGT
I N E D *
vtg4MR2
GTTGATGCAGAACTGTCAGTTTAACTAATATCTGCTAATTGTTGAGATGTTTGTAACATG

961
1021

ATGCTAATAAATTCCCTGCAAGTGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAA

841
281

Fig. 2-4. Nucleotide and deduced amino acid sequences of vtg4 cDNA clone, A391. The stop codon is
represented by an asterisk and the polyadenylation signal, AATAAA, is underlined. Primer sequences
for vtg4MF1/vtg4MR2 (for mapping PCR) and a 3’ end AseI site were underlined.

45

Chapter 2. Zebrafish vtg family
1
1

CTAAAATCTCCTGCTGCAGAAGAAAGTGTTGACCGTATCACAAAAATTAAGCGTACCCTAAAGGCACTCACTAACTGGAAGGCCCTGCCAACTGACCGG
L K S P A A E E S V D R I T K I K R T L K A L T N W K A L P T D R

100
34

CCACTTGCTTCAGCCTATTTCAAAGTATTTGGACAAGAAGTGGCTTATGTCAACTTTGACAAGAGCATCATCGAAGAAGCAATACCGATGGTTACTGGA
P L A S A Y F K V F G Q E V A Y V N F D K S I I E E A I P M V T G

199
67

CCCAAACCACGTGCACTGCTGAAGGAGGCTCTTAAAGCTTTGCAGGAAGGAATTGCATTCCAGTATGCTAAACCTCTGCTTGCAGCTGAAGTGCGTCGT
P K P R A L L K E A L K A L Q E G I A F Q Y A K P L L A A E V R R

298
100

ATTTTCCCAACTGCAGTTGGTGTGCCCATGGAGTTCAGTTGGTACACTGCTGCCGTTGCTGCTGCAAGTGTCAATGTTCAGGCCACCATTACACCTGCT
I F P T A V G V P M E F S W Y T A A V A A A S V N V Q A T I T P A

397
133

CTCCCTGAGAAATTGGAGTCCATGACTTATGAGCAACTAAAGAAGACTGATGTTCAGTTCCAAGCTGAAGCTAGACCAAGTGTTGCTCTCCAGACATTT

L P E K L E S M T Y E Q L K K T D V Q F Q A E A R P S V A L Q T F

496
166

GCTGTGATGGGAGTTAACACTGCCTTCATCCAAGCTGCTGTTATGGCAAGAGGAAAGATCCGTACAATTGCCCCCGGAAAAGTGGCAGCAAGAGCAGAC
A V M G V N T A F I Q A A V M A R G K I R T I A P G K V A A R A D

595
199

ATTCTCAAGGGCAACTACAAGGTGGAGGCTCTGCCTGTTGAACTTCCTGAACACATTGCTTCTGCAAGCTTTGAGACTCTTGCCGTGGTCAGAAACATT
I L K G N Y K V E A L P V E L P E H I A S A S F E T L A V V R N I

694
232

GAAAATCACAGTGCTGAAAGGTCTGTTCCCTTTGTACCTGAATTGTCTCTGCAAANCTCCCAGNCATCTTATGCTGGTGATTTGTCCTCTGAGGTGTCA
E N H S A E R S V P F V P E L S L Q X S Q X S Y A G D L S S E V S

793
265

CCTGATGCTTCAGTGAGAGCTCCTGCTCCATTTGACAGGACCCTCTGTTATGCTGTCCCATACATTGAAATCAAGGGATGTGTTGAGGTGCACTCTCAC
P D A S V R A P A P F D R T L C Y A V P Y I E I K G C V E V H S H

892
298

AATGCTGCTTTTATCAGAAATTCCACTCTTTTCAACATAATTGGACACCACACAGCCCGTGCTGCCATTTCAAGAGCTGAAGGTCCTGCAGTTGAAAGA

N A A F I R N S T L F N I I G H H T A R A A I S R A E G P A V E R

991
331

CTGGAGTTTGAAGTCCAAGTTGGTCCTAGAGCTGCTGAGAGGCTTGTTAAGCAAATCAACATCATTGATGATGATACTCCAGAAGGACAGGCTTTCTTG
L E F E V Q V G P R A A E R L V K Q I N I I D D D T P E G Q A F L

1090 TTGAAACTGAGGGAAATCCTGGACACTGAAGCTAAAAATACACCCGTCTCTTCTGAAAGCAGCAGCAGCAGTCGCAACAGCCGCAGCAGCAGCAGTAGC
364
L K L R E I L D T E A K N T P V S S E S S S S S R N S R S S S S S
1189 AGCACCAGCACCAGCACTAGCAGCAGCAGCAGCAGCAGCAGCAGCTCAAGCTCCTCGTTGTCCAGCTCTCGTATGTCTAAGACTGCCACCATCATAGAG
397
S T S T S T S S S S S S S S S S S S S L S S S R M S K T A T I I E
1288 CCTTTCAGAAAATTCCACAAAGATCGGTACTTGGCACACCATAGTGCCACAAAGGATACTAGCAGTGGAAGCGCGGCAGCTAGCTTTGAGCAAATGCAG
430
P F R K F H K D R Y L A H H S A T K D T S S G S A A A S F E Q M Q
1387 AAAAAGAATAGATTCCTTGGAAATGATATTCCACCTGTTTTTGCTATCATCGCCCGAGCTGTTAGAGCTGACCAGAAGCTTCTGGGCTACCAACTGGCT
463
K K N R F L G N D I P P V F A I I A R A V R A D Q K L L G Y Q L A
1486 GCTTACTTTGACAAACCAACTGCAAGAGTGCAACTCATAGCTTCCTCCATTGCTGAAAATGACAACATGAAGATCTGTGCTGATGGTGCTCTGCTGAGC
496
A Y F D K P T A R V Q L I A S S I A E N D N M K I C A D G A L L S
1585 AAGCACAAAGTCACTGGCAAGTTTTCTTGGGGTGCGGAGTGCAAACAGTATGCAGTCTTTGCTAAAGCTGAAGCTGGTGTCCTGGGTGAATACCCTGCT
529
K H K V T G K F S W G A E C K Q Y A V F A K A E A G V L G E Y P A
1684 GCACGTCTAGAAGTGGAATGGGAGAGACTGCCAAAAATTGTCACCACTTATGCTAAAAAGGTGTGTAAACACATCCTTAATGCAGCTTATAACACAGGA
562
A R L E V E W E R L P K I V T T Y A K K V C K H I L N A A Y N T G
vtg5MF1

1783 TTCAGGTTTGAAAAAGCAACAAACAGCGAGAAAGAGATTGAACTGACTGCAGCCTTGCCATCTCAGAAGTCCTTGAATTTCATTGCTAGGATTCCAGAG
595
F R F E K A T N S E K E I E L T A A L P S Q K S L N F I A R I P E
BamHI
1882 ATCACAATGTCAAAAAGGGATATTCATCTCCCCATCACTGTTCCCATCAATCCAGATGGAACTTTTTCCATTAATGAGGACTTTCTCTCCTGGATCCAT
628
I T M S K R D I H L P I T V P I N P D G T F S I N E D F L S W I H
1981 AAGCATATCATGGAGGAATGAAGAATGAATTACATTCTACATTTTCAAAAGAAAATATGAGCTGTGTTCATGTGTGATGAATGTTTAATTGAATCCTTG
661
K H I M E E *
vtg5MR1
2080 AACATAATGTAAATACAATAAACATCCTTTCAGCAAGTAAAAAAAAAAAAAAAAAAAAA

Fig. 2-5. Nucleotide and deduced amino acid sequences of vtg5 cDNA clone, A227. The stop codon is
represented by an asterisk and the polyadenylation signal, AATAAA, is underlined. Primer sequences
for vtg5MF1/vtg5MR1 (for mapping PCR) and a 3’ end BamHI site were underlined.

46


Chapter 2. Zebrafish vtg family

1
1

GTTGCAGTGGCAAGAGCTGAAGGTCCTGCAGTTGAAAGGCTGGAGTTTGAAGTTCAAGTTGGTCCTAGAGCTGCTGAGAGGCTTGTTAAA
V A V A R A E G P A V E R L E F E V Q V G P R A A E R L V K

91
31

CAAATCAACATCATTGATGACGATACTCCTGAAGGACAGGCTTTCTTGTTGAAACTGAGGGAAATCCTGGACACTGAAGCTAAAAATGCA
Q I N I I D D D T P E G Q A F L L K L R E I L D T E A K N A

181
61

CCCGTCTCTTCTGAAAGCAGCAGCAGTAGCCGCAACAGCCGCAGCATCAGCAGCTCAAGCTCAAACTCAAATTCAAATTCAAGCTCAAGC
P V S S E S S S S S R N S R S I S S S S S N S N S N S S S S

271
91

TCAAGTTCAAGTTCAAGCTCAAGTTCAAGTTCAAGCTCAAGCTCCTCCATGTCCAGCTCTCGTATGTCTAAGACTGCCACCATCATGGAG
S S S S S S S S S S S S S S S S M S S S R M S K T A T I M E

361
121

CCTTTCAGGAAATTTCACAAAGACCGGTACTTGGCAGCACATAGTGCCACAAAGGATACTAGCAGCGGAAGTGCTGCAGCTAGCTTTGAA
P F R K F H K D R Y L A A H S A T K D T S S G S A A A S F E

451
151

CAAATGCAGAAAAAGAATAGATTCCTTGGAAATGATATTCCACCTGTTTTTGCTATCATCGCCCGTGCTGTTAGAGCTGACCAGAAGCTT
Q M Q K K N R F L G N D I P P V F A I I A R A V R A D Q K L

541
181

CTGGGCTACCAACTGGCTGCTTACTTTGACAAACCAACTGCAAGAGTGCAACTCATAGCTTCCTCCATTGCTGAAAATGACAACATGAAG
L G Y Q L A A Y F D K P T A R V Q L I A S S I A E N D N M K

631
211

ATCTGTGCTGATGGTGCTCTGCTGAGCAAGCACAAAGTCACTGCCAAGTTTTCTTGGGGTGCGGAGTGCAAACAGTATGCAGTCTTTGCT
I C A D G A L L S K H K V T A K F S W G A E C K Q Y A V F A

721
241

AAAGCTGAAGCTGGTGTCCTGGGTGAATACCCTGCTGCACGTCTAGAGGTGGAATGGGAGAGACTGCCAAAAATTGTCACCACCTATGCT
K A E A G V L G E Y P A A R L E V E W E R L P K I V T T Y A

811
271

AAAAAGCTTTCTAAATGCATACTTAATGCAGCTTATGACACAGGATTCAGGTTTGAAAAAGCAACAAACAGCGAGAAAGAGATTGAACTG
K K L S K C I L N A A Y D T G F R F E K A T N S E K E I E L

901
301
991
331

ACTGCAGCCTTGCCATCTCAGAGGTCCTTGAATGTCATTGCTAGGATTCCAGAGATCACAATGTCAAAAAGGGATATTTACCTCCCCGTC
T A A L P S Q R S L N V I A R I P E I T M S K R D I Y L P V
AseI

ACTGTTCCCATCAATCCAGATGGAACTTTTTCCATTAATGAGGACTTTCTTTCCTGGATTCACAAGTATATTAAGGAGGATTGAGGAATG
T V P I N P D G T F S I N E D F L S W I H K Y I K E D *

1081

AATTGCAATCTCCATTTTCAAAAGAAAATAGGAGCTGTGTTAATGCATAACTGTCAGTTTAATCAATAATATTTTGTAATGTGATGCTAA

1171

TAAATACCCTGCAAGTGTAAAAAAAAAAAAAAAAAA

Fig. 2-6. Nucleotide and deduced amino acid sequences of vtg6 cDNA clone, A220. The stop codon is
represented by an asterisk. The polyadenylation signal, AATAAA, and a 3’ end AseI site were
underlined.

47


Chapter 2. Zebrafish vtg family
1
1
61
21

TGGAAGTGCTGCAGCTAGCTTTGAGCAAATGCAGAAACAGAATAGATTCCTTGGAAATGA
G S A A A S F E Q M Q K Q N R F L G N D
vtg7MF2
TCTTCCACCTGTTTTTGCTATCATCGCCCGTGCTGTTAGAGATGACCAGAAGCTTCTGGG
L P P V F A I I A R A V R D D Q K L L G

121
41

CTACCAACTCGCTGCTTACTTTGACAAACCAACTGCAAGAGTGCAACTGATAGCTTCCTC
Y Q L A A Y F D K P T A R V Q L I A S S

181
61

TATTGCTGAAAATGACAACAGGAAGATCTGTGCCGATGGTGCACTACTGAGCAAGCACAA
I A E N D N R K I C A D G A L L S K H K

241
81

AGTCACTGGCAAGTTTTCTTGGGGTGCGGAGTGCAAACAGTATGCAGTCTTTGCTAAAGC
V T G K F S W G A E C K Q Y A V F A K A

301
101

TGAAGCTGGTGTCCTGGGTGAATTCCCTGCTGCACGTCTAGAAGTGGAATGGGAGAGACT
E A G V L G E F P A A R L E V E W E R L

361
121

GCCAATAATTGTCACCACCTATGCCAAAAAGCTGTGTAAGCACATCCTTAAGGCAGCATA
P I I V T T Y A K K L C K H I L K A A Y

421
141

TGACACGGGATTCAGGTTTGAAAGAGCAACAAACAGCGAGAAAGAGATTGAACTGACTGC
D T G F R F E R A T N S E K E I E L T A

481
161

AGCCTTGCCATCTCAGAAGTCCTTTGATTTCATTGCTAGGATTCCAGAGATCACAATGTC
A L P S Q K S F D F I A R I P E I T M S

541
181
601
201

AAAAAGAGAGATTCCTCTGCCAGTTGCTGTTCCCATCAATCCAGACGGAACTTTTTCCAT
K R E I P L P V A V P I N P D G T F S I
BamHI
TCATACTTATGAGGACTTTCTCTCCTGGATCCAGAAATACATCAAGGATGAATAAGAAAT
H T Y E D F L S W I Q K Y I K D E *

661

TATTTATTATCTTCATTTTCACAAGAATGTTTGAAGATAATTATATTTGAATATTTTTTT

721

AGTGATGTAAACAAAATAAACGTCCTCTCTGCAAGTATAAAAAAAAAAAAAAAAAAA

vtg7MR1

Fig. 2-7. Nucleotide and deduced amino acid sequences of vtg7 cDNA clone, A349. The stop codon is
represented by an asterisk and the polyadenylation signal, AATAAA, is underlined in italic letters.
Primer sequences for vtg7MF2/vtg7MR1 (for mapping PCR) and a 3’ end BamHI site were underlined.

48


Chapter 2. Zebrafish vtg family
a 5’ end 1802-bp sequence was obtained and the resulting near full-length vtg3 cDNA is
3938 bp long (excluding poly-A tail), encoding a Vtg3 with 1251 amino acid residues
(Fig. 2-3). Retrieval of the missing 5’-end cDNA sequences for the other five vtg cDNA
clones has not been carried out. The insert lengths for vtg2 and vtg4-7 cDNA clones range
from 777 to 2138 bp, encoding partial Vtgs with 217 to 666 amino acid residues (Figs. 22, 2-4 to 2-7). After multiple sequence alignment of the seven vtg cDNAs, it was revealed
that cDNA clone A183 (vtg2) has a 771-bp extension of 3’ end coding region and may
represent a unique vtg. The heterogeneity of vtg cDNAs may be a common phenomenon
in teleost fish since similar observations were reported previously in other species. For
example, vtg cDNA clones pSG Vg 5.50 from rainbow trout (Oncorhynchus mykiss) and
pOA Vg 71 from tilapia (Oreochromis aureus) were found have 1.5-kb and 400-bp
extension towards the 3’ end respectively when compared with other vtg cDNA clones in
the same fish (Le Guellec et al., 1988; Ding et al., 1990). It is worth to note that a “CGLC
motif”, which was believed to be required for protein multimer assembly (Mayadas and
Wagner, 1992) was found in the extension portion of the deduced Vtg2 amino acid
sequence as well as in the similar position of rainbow trout Vtg1 (Figs. 2-2 and 2-9).
However, no such motif was found in either the full-length Vtg1 or Vtg3 sequences. It is
also noteworthy that the lengths of the 3’ UTRs for vtg1 and vtg4-7 are very similar
(ranging from 103 to 129 bp)in contrast with that of vtg2 (84 bp) or vtg3 (180 bp) (Figs. 21 to 2-7).

2.3.1.2. Sequence comparison of the zebrafish Vtgs

Multiple alignment of the C-terminal deduced amino acid sequences of Vtg1-7 showed
that there is a high degree of sequence similarity between most members except for Vtg2
49


Chapter 2. Zebrafish vtg family
and Vtg3. As shown in Fig. 2-8, about 84% of the C-terminal 215-217 amino acid residues
are identical in Vtg1 and Vtg4-7. Among the 35 sites with different amino acid residues in
different Vtgs, 30 of them were replaced by highly similar or similar amino acid residues.
Only 5 of them were substituted by dissimilar amino acid residues (Fig. 2-8). Thus, it is
reasonable to conclude that vtg1 and vtg4-7 might arise from recent duplications of a vtg1like precursor gene, while vtg2 and vtg3 probably arose from more ancient gene
amplification event.

All known vertebrate Vtgs can be divided into three common domains, LVI, PV and LVII
based on precursor-product relationships between Vtg and yolk protein products (LaFleur,
1999). In addition, in the entire Vtg sequence, five subdomains (I-V) have been defined
which can be aligned relatively easily among Vtgs of divergent species from invertebrates
to oviparous vertebrates (Chen et al., 1997). By sequence comparison with rainbow trout
Vtg1 (Mouchel et al., 1996), the lengths of the three domains in zebrafish Vtg1 are similar
to those of corresponding domains in rainbow trout Vtg1 except for LVII which is 278
amino acids shorter in the zebrafish Vtg1 (Fig. 2-9). Based on the definition by Chen et al.
(1997), zebrafish Vtg1 contains only homologous subdomains I-III, but lacks IV and V
(Fig. 2-9). Deduced from our partial EST clone A183, zebrafish Vtg2 contains a truncated
LVI domain but intact domains of PV and LVII (Fig. 2-9). Interestingly, zebrafish Vtg2 is
unique in that the length of its LVII domain (comparable to that in rainbow trout Vtg1) is
the longest among those of the seven Vtgs. In contrast to Vtg1, zebrafish Vtg2 contains
homologous subdomains IV and V. As for zebrafish Vtg3, it contains LVI and LVII, but
no PV domain (Fig. 2-9). In addition, the LVII domain in Vtg3 is the shortest among the
seven Vtgs. Similar to Vtg1, zebrafish Vtg3 contains only subdomains I-III.
50

Chapter 2. Zebrafish vtg family

Vtg1
Vtg2
Vtg3
Vtg4
Vtg5
Vtg6
Vtg7

SRMSKTATIIEPFRKFHKDRYLAHHSATKDTSSGSAAASFEQMQKQNRFLGNDIPPVFAIIARAVRADQKLLGYQLAAYF 1190
--w---l-km-a-------q-kt--gds-..--r-tgs-l--i---s-y---tv------------v-r------fv-f- 182
lnqrvfkekrdentsceerktssslpv-q-...................ldvtpd--vtvk-lslspqa-p---egv-fy 1092
---------m-------------p---------------------k---------------------------------- 116
---------------------------------------------k---------------------------------- 498
---------m-------------a---------------------k---------------------------------- 189
.................................--------------------l------------d------------47
†
‡
†

Vtg1
Vtg2
Vtg3
Vtg4
Vtg5
Vtg6
Vtg7

DKPTARVQL...IVSSIAENDNMKICADGALLSKHKVTGKFS.WGAECKQYAVFAKAEAGVLGEFPAARLEVEWER.LPI 1265
---ss----...-a--------f-f----v--------s-vt.------e---tt-----l------f---w----.--- 257
l.---qkddiem---evg-ea-w-m--nahfdrt-tsakahlr-----qt-d-smrvs-acqp-skpsistkinwgt--s 1171
---------...-a------------------------a---.---------------------y-----------.--k 191
---------...-a----------------------------.---------------------y-----------.--k 573
---------...-a------------------------a---.---------------------y-----------.--k 264
---------...-a--------r-------------------.---------------------------------.--- 122
†
†
‡
‡
#

Vtg1
Vtg2
Vtg3
Vtg4
Vtg5
Vtg6
Vtg7

IVTTYAKKLGKHILTAAYDTGFRFERATNSEKEIELTAALPSQRSLNIIARIPEITMSKRDIYLPVAVPINPDGTFSIET 1345
-f-------s---pm--lqa--nv---k-----l---v----k-t--v-v-v--m---rm--p---t---------dvhf 337
vf--vgqivqeyvpgvs-im--yqknee-p-rqasv-vvas-petfdlkvk---r-iy-kk-ps-ielvgieaanltms- 1251
-as-----vs-y--n--------------------------f-k---v----------r-------t---------t-d. 270
--------vc----n---n------k-----------------k---f--------------h--it-----------n. 652
---------s-c--n----------k---------------------v------------------t-----------n. 343
---------c----k----------------------------k-fdf------------e-p---------------h- 202
††

‡† # †
‡
‡
# ‡ ‡‡‡
‡ ‡ # ‡†
† †

Vtg1
Vtg2
Vtg3
Vtg4
Vtg5
Vtg6
Vtg7

YEDFLAWIQKYIKEE*................................................................ 1360
---iyfra-n--ydyttaqcsmmqdtistfnnktyknempiscyqvlaqdctselkfvallkkdeesekthlnvklvdid 417
*............................................................................... 1251
.k---s--p---n-d*................................................................ 284
.----s--h-h-m--*................................................................ 666
.----s--h-----d*................................................................ 357
-----s-------d-*................................................................ 217
†
† † # †‡‡

Fig. 2-8. Multiple alignment of the C-terminal deduced amino acid sequences of Vtg1-7 by Clustal W.
Dashes represent identical amino acid residues as in Vtg1. Dots are inserted for maximum alignment.
Asterisks mark the C-terminal ends of Vtgs. The degree of similarity for the substituted amino acid
residues in Vtg1 and Vtg4-7 is indicated below the Vtg7 sequence. ‡, highly similar; †, similar; #,
dissimilar. Two vertical lines define the comparison region. Sequences in the aligned region marked by

horizontal lines were used in phylogenetic analysis (see Fig. 2-10).

51