Nucleotide sequences of the mouse globin beta gene cDNAs in a wild
derived new haplotype Hbb
Laboratory of Animal Cell Technology, Fukuyama University, Fukuyama, Hiroshima-ken, 729-0292, Japan
Experimental Animal Center, Kagawa Medical University, Miki-cho, Kagawa-ken, 761-0793, Japan
Department of Physiology, Osaka University Medical School, Suita, Osaka, 565-0871, Japan
Mammalian Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka-ken, 411-8540, Japan
Lanzhou Institute of Biological Products, Lanzhou, China
The Graduate University for Advanced Studies, Hayama, Kanagawa-ken, 240-0193, Japan
Received: 5 January 1999 / Accepted: 5 April 1999
Abstract. Haplotypes of the beta-globin gene complex (Hbb)in
laboratory mice have been defined as d, p, and s. We previously
found a new haplotype w1 in wild mice collected from northwest-
ern China. This study analyzed the nucleotide sequences of b1 and
b2-globin gene cDNAs of both the p and w1 haplotypes, in com-
parison with those of the d haplotype. In Hbb-b1 cDNA, six base
substitutions were found between the d and w1 haplotypes and also
between p and w1, but none existed between d and p. In Hbb-b2
cDNA, three base substitutions were found between the d and w1
haplotypes and also between d and p, but none between p and w1.
This result indicated that the Hbb gene complex of the p haplotype
genes and is probably a recombinant between
d and w1 haplotypes. The hemoglobin containing the W1 pheno-
type showed oxygen-binding properties identical with those of the
hemoglobins containing D and P phenotypes.
Three kinds of beta hemoglobin haplotype d, p, and s have been
reported in laboratory mice (Staats 1980). Almost all laboratory
mice have either the s or d haplotype, except AU/SsJ, which car-
ries the p haplotype (Staats 1980). In wild mouse populations,
extensive geographical surveys of the genetic polymorphism of
hemoglobin have been carried out both in Europe (Bonhomme et
al. 1989) and Asia (Minezawa et al. 1979; Miyashita et al. 1985;
Kawashima et al. 1991, 1995). Recently, we found a new haplo-
type, w1, that was observed mainly in northwestern China. It can
be clearly distinguished from the other known haplotypes on cel-
lulose acetate membrane electrophoresis after treatment with
monoiode-acetate (Kawashima et al. 1991).
This study attempts to determine cDNA nucleotide sequence
variations in Hbb-b1 and Hbb-b2 genes of w1 and p haplotypes, in
comparison with the previously reported sequences of the d hap-
lotype (Konkel et al. 1979).
We also compared the oxygen-binding properties of W1 he-
moglobin with those of D and P hemoglobins, since the altitude in
northwestern China, where the w1 hemoglobin mice were found, is
Materials and methods
Mice with the p haplotype from which the new inbred strain, MSM,
has been developed were captured at Mishima in 1978. Mice with the w1
haplotype were captured at Jiayuguang, Gansu Province in China in 1981.
BALB/cAnN congenic strains carrying either Hbb
of the MSM strain or
of the Gansu mouse have been established by repeated backcrosses
of eight generations in the former and five in the latter case. The congenic
thus developed have been main-
tained in the National Institute of Genetics. BALB/cAnN mice were pur-
chased from SLC Inc., Japan. AU/SsJ strain was purchased from The
Jackson Laboratory (Bar Harbor, Me., USA) and has been maintained in
the National Institute of Genetics.
Isolation of mRNA.
After repeated injections of acetylphenylhydrazine
into adult female mice of each strain at 4 mg/100 g body weight/day for 6
days, blood was drawn from the posterior vena cava (Borsook et al. 1952).
Reticulocytes were collected by centrifugation at 8000 rpm for 5 min, and
mRNA was isolated with an mRNA purification kit (Pharmacia Biotech ).
Determination of cDNA sequences of Hbb-b1 and Hbb-b2.
cDNAs were synthesized from isolated mRNA with a cDNA synthesis kit
(Gibco/BRL ). Templates for sequencing were the PCR product amplified
from the cDNAs with primer sets of forward (F) 5Ј-CAC-ATT-TGC-TTC-
TGA-CAT-A-3Ј at starting position 180 and reverse (R) 5Ј-TTT-TTA-
TTT-GTC-AGA-AGA-CAG-ATT-TT-3Ј at starting position 1533 for
Hbb-b1 gene, and (F) 5Ј-TAC-GTT-TGC-TTC-TGA-GTC-T-3Ј at starting
position 222 and (R) 5Ј-CAT-TTA-TTA-GAT-AAA-AGC-TAG-ATG-
CC-3Ј at starting position 1550 for Hbb-b2 gene based on the sequence
data of genomic DNA of Hbb
in the BALB/c mouse (Konkel et al. 1979).
Sampled cDNAs were amplified with 1.5 m
, 100 ng cDNA, 20
pmol of each primer, 0.25 m
dNTP, and 1.25 units of Taq DNA poly-
merase (TaKaRa Ex TaqTM kit) in 50 ml per sample. The samples were
pre-incubated for 10 min at 94°C. Then, 25 cycles of amplification were
performed for 1 min at 94°C for denaturation, for 1 min at 55°C for
annealling, and for 1 min at 72°C for extension. Thereafter, the samples
were incubated for 10 min at 72°C. Amplified DNA fragments were sepa-
rated by electrophoresis with 2% agarose gel and detected by ethidium
bromide staining. Sequencing by the dideoxy method was performed with
a sequencing kit and DNA sequencer, Model 373 (ABI).
Oxygen-binding properties of red blood cells and hemoglobin.
Blood samples were collected with a heparinized syringe from the hearts of
adult mice. Concentration of red cell 2,3-diphosphoglycerate (2,3-DPG)
was determined with an enzymic assay kit (35-A, Sigma ). Red cell sus-
pensions for oxygen-binding experiments were prepared by diluting the
blood sample with a 150-fold volume of isotonic phosphate buffer (0.15
, pH 7.45). Hemoglobin solutions (whole he-
molysates) were prepared by washing red cells three times with large
volumes of 0.9% NaCl and hemolyzing with one volume of 0.05
buffer (pH 7.4), followed by centrifugation at 10,000 rpm for 10 min. The
whole procedure for hemolysate preparation was conducted at 4°C. The
Correspondence to: K. Moriwaki
Mammalian Genome 10, 879–882 (1999).
© Springer-Verlag New York Inc. 1999