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https://www.abbs.info/ e-mail:[email protected] ISSN 0582-9879 |
A High-resolution Genetic and Physical
Map of a Mouse Coat Abnormity Locus (Uncv)
SHI Yao-Zhou1,2,
WANG Dong-Ping3, LIU Tao-Tao2,
TENG Xiao-Kun2, WANG Zhi-Qiang2, HU Lan-Dian2,
LI Ji-Qing2,
ZHAO Guo-Ping2, WANG Yu-Zhen, KONG Xiang-Yin2*,
LI Shan-Ru3*
(
1 School of Life Science, University of Science and Technology of China, Hefei
230027, China ;
2 Research Center of Biotechnology, Shanghai Institutes for Biological Science,
the Chinese Academy of Sciences,
Shanghai 200233, China; 3 Institute of Jingfeng Medical Laboratory Animals,
Beijing 100071, China )
Abstract
More than a hundred of loci (genes) affect the development of mouse and human
hair. A locus of Uncv(uncovered) has been confirmed to be involved
in hairlessness for homozygote and sparse hair for heterozygote. Except hairlessness(or
called uncovered coat), the homozygote was also accompanied by growth retard
and puberty delay. Identification of the mutation in the gene will be important
for understanding the related diseases in human. Although the uncovered locus
(Uncv) has been mapped to the mouse distal chromosome 11(Chr11), the
high-resolution genetic map and physical map of the locus has not been created.
In this study, 2074 F2 mouse populations from backcross [BALB/c (Uncv/Uncv)
× C3H (+/+)]× BALB/c (Uncv/Uncv ) and [BALB/c (Uncv/Uncv
)× C57BL/6 (+/+)] × BALB/c(Uncv/Uncv) were genotyped using 16
polymorphic markers with an ≈20 cM interval on mouse distal Chr11. By genetic
linkage analysis, Uncv locus was mapped to an ≈1.4 cM interval between
markers D11Mit337 and D11Mit338 with the following order: proximal D11Mit338-D11Mit203
(Uncv)-D11Mit103 -D11Mit337 distal on mouse Chr11. And then, a contig
of 35 BACs representing the Uncv– containing region was constructed.
The contig covered 800－1000 kb region flanked by 189K10-SP6 and D11Mit103.
Together, we have constructed the high-resolution genetic map and detailed
physical map of the Uncv region. This will facilitate the identification
of the Uncv loci.
Key words mouse;
hair; mutation; genetic map; physical map
Hair development, which
includes hair follicle morphogenesis, matrix cells proliferation differentiation,
and destruction[1], is a very complex progress called hair cycle. It is subject
to the influence of many genes. In man, more than 300 genetic conditions cause
hair abnormalities as a component feature[2]. In mouse, more than 100 mutations
affect its coat texture and structure[3], each caused unique phenotypic and
genetic characteristics, such as bald arthritic (Bda, Chr11)[4], depilated
(dep, Chr4)[5], forkhead box N1 (Foxn1, Chr11)[6], hairless (hr, Chr14)[7],
waved alopecia (Wal, Chr14)[8]. By positional cloning and screening for phenotype
similarities with genetically engineered mice, researchers have got the molecular
characteristics of some mutants like hr[9], Foxn1[6], wa-1[10] and fs[11].
But most of the mutant genes have remained unidentified.
The uncovered (Uncv, MGI:70615) coat mutation appeared spontaneously
in BALB/c background. It inherited an autosomal semidominant (homozygote with
uncovered coat, heterozygote with sparse hair). Except uncovered coat, the
homozygote was also accompanied by growth retard and puberty delay. Although
Uncv has been located on the distal chromosome 11[12], its high-resolution
genetic map and physical map have not been determined.
In this study, we first established the high-resolution genetic map by genotyping
2074 intercross-backcross F2 progeny with 16 polymorphic markers flanking
this locus. After the Uncv locus was precisely mapped to an interval
between markers D11Mit337 and D11Mit338, we constructed a BAC-based contig
map. Although we have not identified the candidate gene responsible to the
mutation, the establishment of the high-resolution genetic map and physical
map will facilitate the cloning of Uncv locus.
1 Materials and Methods
1.1 Animals
All mice including the mutant inbred strain BALB/c (Uncv/Uncv)
were obtained from the Institute of Jingfeng Medical Laboratory Animals and
maintained at the Intitute under the standard lab conditions.
According to the polymorphism of the markers used in the study, we set up
two intercrosses. First, the intercross was made between BALB/c (Uncv/Uncv)
and C3H, then the F1 mice were backcrossed to BALB/c (Uncv/Uncv).
218 F2 progeny from this intercross-backcross were genotyped using 10 polymorphic
markers D11Mit132, D11Mit199, D11Mit360, D11Mit180, D11Mit202, D11Mit214,
D11Mit338, D11Mit103, D11Mit48 and D11Mit184.
Second, according to the polymorphism of the markers around the D11Mit338,
the other intercross-backcross, [BALB/c (Uncv/Uncv) × C57BL/6]
× BALB/c (Uncv/Uncv) was carried out. 1856 F2 backcrossed mice
were tested for further mapping using 6 markers, D11Mit256, D11Mit338, 189K10-SP6
(see below ), D11Mit203, D11Mit 337 and D11Mit104.
1.2 Phenotyping
All these F2 mice were phenotyped at 1－3 weeks of age when they were easily
recognized as uncovered coat and sparse coat.
1.3 Genotyping
Mouse genomic DNA was isolated from liver tissue following the standard lysis
protocol. PCR amplications were performed on a PTC-225 DNA Engine Tetrad (MJ
Research Co.), the reactions were carried out with a touch-down program in
a 10 μl mixture containing 50 ng genomic DNA, 2.0 mmol/L of each dNTP, 1.0
pmol of each M13-tailed primer, 1.0 pmol of IRD-labeled M13 primer (Li-cor
Co.), 1.5 mmol/L MgCl2, 1× PCR reaction buffer and 0.25 unit of AmpliTaq Gold
DNA polymerase (Perkin-Elmer Co.). The thermal cycle consisted of 95°C for
10 min, followed by the first 9 cycles of denatured at 95°C for 30 s, annealing
at 68°C for 2 min, 8-cycle drop 1°C per cycle to 52°C and extension at 72°C
for 1 min followed by an additional 25 cycles of denaturation at 95°C for
30 s, annealing at 54°C for 2 min and extension at 72°C for 1 min. Finally
the mixture was extended at 68°C for 1 h.
The PCR products labeled by M13 IRD700/IRD800 fluorescent primers were electrophoresed
on Li-cor DNA4200 sequencer using 7% PAGE and 7 mol/L urea gels following
the standard protocols described in the user’s manual of the machine, the
fluorescent dye labeled DNA markers were loaded simultaneously and the data
were collected and analyzed with BaseImage 4.1 and RFLPscan software[13].
A M13-tail was added to the 5′ end of one of a primer used for genotyping,
so that the M13 IRD700/IRD800 fluorescent primer could bind to it. The primer
sequences were obtained from the Mouse Genome Database (MGD, http://www.informatics.jax.org).
1.4 BAC library screening and construction of the physical map
The RPCI-23 female (C57BL/6J) mouse BAC library (Research Genetics Co.) was
screened by hybridization[14] with radioactively probes[15] . The library
was screened twice, first with the probe generated from D11Mit203,, second
with pooled probes from the end sequences of positive BAC clones for chromosome
walking. The secondary screening results were confirmed by PCR. All probes
were labeled with [α-33P] dCTP, The SP6 and T7 ends of BAC clones were sequenced
on a ABI377 machine following the standard protocol from Washington Sequence
Center. All the end sequences were screened for repeptitive elements by RepeatMasker
(http: //repeatmasker.genome.Washington.edu), and non-repetitive se-quences
were used as probes. Closure of this contig was accomplished by bi-directional
walking with the end sequences of the BAC clones. Isolation of new polymorphic
markers and refine mapping were performed at the time.
2 Results
2.1 Genetic map of the Uncv region
The Uncv locus was previously mapped to the interval between Es3 and
D11Mit8 on mouse distal chromosome 11. To localize it more precisely, 218
F2 mice from (BALB/c (Uncv/Uncv) × C3H) × BALB/c (Uncv/Uncv)
backcross were analyzed. After the F2 progeny were classified into two groups:
uncovered and sparse coats, these animals were genotyped by using 10 closely
linked genetic markers between D11Mit132 and D11Mit184 covering ≈20 cM interval
(http: //www.jax.org).
Fig.1 Genetic mapping of Uncv critical region
(A) Order and haplotypes for markers on Chr11, the loci are listed centromeric
to telomeric from top to bottom. Each column represents an observed haplotype,
data are shown for the 134 (out of 218) F2 backcross mice exhibiting recombination
between markers in the interval between D11Mit132 and D11Mit184, totally 218
mice [BALB/c(Uncv/Uncv)× C3H] × BALB/c(Uncv/Uncv)
were genotyped. The number of backcross progeny inheriting each haplotype
is depicted below each column. Filled squares represent recombination at the
named locus, including homozygosity for BALB/c allele in hairless phenotype
and heterozygosity of BALB/c and C3H allele in sparse phenotype. Empty squares
represent nonrecombination at the named locus, including homozygosity in sparse
mice and heterozygosity in hairless mice.
(B) haplotype analysis of backcross 2 [BALB/c(Uncv/Uncv) × C57]
× BALB/c(Uncv/Uncv), totally 1856 mice were genotyped and no
mouse was recombined at D11Mit203, 22 animals showed recombination at D11Mit337,
6 mice recombinated at D11Mit338 and the new polymorphic marker 189K10-SP6,
all recombinant animals at D11Mit337 were verified at D11Mit104 which located
at the telomeric region, all recombinant animals at D11Mit338 were verified
at D11Mit256 which was centromeric to D11Mit338.
Of the 218 mice tested,
134 mice exhibited recombinations between markers in the interval [Fig.1(A)]
and there were 11 recombinations between Uncv and D11Mit214 (≈5.0 cM),
and 3 recombinations between Uncv and D11Mit103 (≈1.4 cM). So, we determined
that the Uncv locus located in the ≈6.4 cM interval flanked by D11Mit214
and D11Mit103. Based
on the polymorphism of the markers in this interval, the other cross was carried
out to refine the position of Uncv. 1856 F2 mice from [BALB/c (Uncv/Uncv) ×
C57BL/6] × BALB/c (Uncv/Uncv) were genotyped using 5 known polymorphic
DNA markers and a novel polymorphic microsatellite, 189K10-SP6. It was the
SP6 end sequence of the BAC 189K10 which was sequenced and confirmed polymorphism
between BABL/c and C57BL/6. Of these tested markers, there was no recombination
between D11Mit203 and Uncv locus, 28 recombinations between D11Mit337
and 189K10-SP6, 6 recombinations between Uncv and 189K10-SP6. So, the
Uncv locus was further mapped to ≈1.4 cM region flanked by D11Mit337
in distal and 189K10-SP6 in proximal end of mouse Chr11 [Fig.1(B)].
2.2 Construction of physical map
Because there was no recombination between Uncv and D11Mit203, we first
used D11Mit203 as the probe to screen the RPCI-23 mouse BAC library, and then
used the end sequences from the positive BAC clones for chromosome walking.
We totally used 51 STSs to assemble these BAC clones. Three of these STSs
were polymorphic markers, others were the end sequences of BACs, 14 of which
were directly sequenced in our lab, others were obtained from database. Some
of these BAC end sequences were tested polymorphic between BALB/c and C57BL/6,
and were used as markers to genotype the F2 progeny to reduce the Uncv-containing
critical region. After genotyping 6 recombinants between Uncv and D11Mit338,
we found they all recombined between Uncv and 189K10-SP6. In addition,
D11Mit103 which had recombinations in the first backcross (shown in Fig.1)
was mapped to the several overlapped BACs (Fig.2). Consequently, we constructed
a contig of 35 BACs representing the Uncv-containing region (Fig.2)
which was flanked by 189K10-SP6 and D11Mit103.
3 Discussion
Uncv mutant mouse appeared spontaneously in the production stock of
BALB/c mice with a normal coat. Our previous analyses have shown that mutation
corresponds to a single, autosomal, semidominant gene, and has been mapped
into a 30 cM interval between D11Mit8 and Es3[12] . Although several mutant
genes responsible for skin and coat structure have been mapped on Chr11, this
mutant was confirmed as a new locus affecting skin and coat texture because
of its unique phenotype and chromosomal location.
In the present study, we first localized Uncv locus to an ≈1.4 cM interval
between D11Mit337 and D11Mit338 by genotyping 2074 F2 offspring from two intercross-backcrosses,
and found that D11Mit203 was the closest marker to Uncv. Based on this
mapping data, we tried to construct a BAC contig for further positional cloning.
Fig.2 high-density
physical map of the Uncv locus region, which consists of 35 BAC clones
STS markers and BAC ends which were used to assemble the BACs are listed from
centromere(left) to tetomere(right). The heavy horizontal lines represent
BACs and the vertical lines represent STSs and BAC ends used to PCR identification
of the overgos. Filled cycle indicated that the BAC was anchored by PCR with
the primer pairs used to hybridize or assemble, and no filled cycle in the
end of the clone indicates that the precise end point are unknown. The paired
vertical lines on the left represent a recombination hot spot , as there are
6 recombinant events between 189K10-SP6 and D11Mit203 in our backcross, but
these two markers are all contained on the same BAC clones. On the right of
the contig we have mapped D11Mit103 onto 5 BACs, which is excluded in our
first round of backcross.
By using D11Mit203 and
the pooled BAC end sequences as markers to screen mouse BAC library, we constructed
the contig of 35 BACs representing the Uncv-containing region. Further
genetic linkage analysis showed that the contig was flanked by 189K10-SP6
and D11Mit103 (Fig.2). This newly established physical map suggested that
we have narrowed the Uncv region to 800-1000 kb. It also suggested
that the order of the tested markers within this region could be: proximal
D11Mit338-D11Mit203 (Uncv)-D11Mit103 -D11Mit337 distal on mouse Chr11,
while the Integrated MIT SSLP and Copeland/Jenkins RFLP genetic maps (http:
//www-genome.wi.Mit.edu/cgi-bin/mouse/) show that they are in the same locus
without an established order, and MGI (http: //www. jax.org) maps shows the
order of D11Mit203-D11Mit337-D11Mit338-D11Mit103.
The mouse has been proved to be a powerful model and tool for studies of human
genetic diseases including skin and hair disorders[16]. Although there are
more than 100 mutations affecting mouse skin and hair and many genetically
engineered mice often exhibit various abnormal cutaneous phenotypes, only
some of their genes and biochemical mechanism has been known. Positionally
cloning of Uncv and find it counterpart in human will be important
for understanding and treating some human hair growth disorders. Together,
we have constructed the high-resolution genetic map and detailed physical
map of the Uncv region. This will enforce the identification of the
Uncv loci.
Acknowledgements We thank Dr. Steve Scherer for kindly providing the
BAC clones.
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