Characterization of the CHORI-240 BAC clones containing the bovine CSN1S1, CSN2, STATH, CSN1S2 and CSN3 genes.

Abstract

Nineteen BAC clones were identified by hybridization of the bovine genomic BAC library CHORI-240 with mixed CSN1S1- and CSN3-specific probes. Two of the clones were shown to contain the genes CSN1S1, CSN1S2, CSN2, STATH and CSN3, and five were proved to include the genes CSN2, STATH, CSN1S2 and CSN3. These data showed that the BAC contig was established for the whole casein cluster, including all known five genes.

Full text

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The structure of the casein gene cluster is highly
conserved among mammalian species (Rijnkels
et al. 1997a; Rijnkels et al. 1997b; Rijnkels et al.
2003). In the human, mouse, rat and cattle, these
genes are ordered in the same way: CSN1S1,
CSN2, STATH, CSN1S2 and CSN3 (Rijnkels et al.
1997a;Rijnkelsetal.2003). TheHTN1andHTN3
genes have been localized in human, mouse
and rat casein clusters but still have not been iden-
tifiedincattle.Herewereportonobtainingofcon-
tinuousbovine DNA
the 5’-flanking region and coding part of the bo-
vine casein gene cluster via hybridization of the
bovine genomic BAC library CHORI-240 with
CSN1S1- and CSN3-specific DNA probes.
sequences covering
Polymerase chain reaction (PCR) fragments
representing the 5’-flanking region and exon 1
of bovine gene CSN1S1 (GenBank accession num-
ber X03590) and exon 4 and intron 4 of CSN3
(AJ841946) were amplified from bovine genomic
DNA by using the following primer pairs:
CSN1S1fw,GAAGAA
CSN1S1rv,TGCATGTT
CSN3fw, GAAATCCCTA CCATCAATACC; and
CSN3rv, CCATCTACG CTAGTTTAGATG. PCR
yieldedproductsof319bpfortheCSN1S1geneand
270 bp for CSN3.
Large insert clones containing CSN1S1 and
CSN3, with average insert size of 180 kb, were de-
tected by screening of bovine genomic BAC li-
brary CHORI-240 Segment I (http://bacpac.chori.
GCAGCAAGCTGG;
CTCATAATAACC;
J Appl Genat 47(3), 2006, pp. 243–245
Short communication
Characterization of the CHORI-240 BAC clones containing
the bovine CSN1S1, CSN2, STATH, CSN1S2 and CSN3 genes
Alexei A. Sazanov1, Tadeusz Malewski2, Stanis³aw Kamiñski3, Lech Zwierzchowski2
1Laboratory of Molecular Genome Organization, Institute of Farm Animal Genetics and Breeding, Russian Academy
of Agricultural Sciences, Moskovskoye sh. St Petersburg–Pushkin, Russia.
2Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzêbiec,
Wólka Kosowska, Poland
3Department of Animal Genetics, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
Abstract. Nineteen BAC clones were identified by hybridization of the bovine genomic BAC library
CHORI-240withmixedCSN1S1-andCSN3-specificprobes.Twoofthecloneswereshowntocontainthegenes
CSN1S1, CSN1S2, CSN2, STATH and CSN3, and five were proved to include the genes CSN2, STATH, CSN1S2
and CSN3. These data showed that the BAC contig was established for the whole casein cluster, including all
known five genes.
Key words: BAC-libraries, Bos taurus, contig, casein genes.
Received: November 15, 2005. Revised: January 11, 2006. Accepted: January 23, 2006.
Correspondence: T. Malewski, Department of Molecular Biology, Institute of Genetics and Animal Breeding,
Polish Academy of Sciences, Jastrzêbiec, 05–552 Wólka Kosowska, Poland; e-mail: t.malewski@ighz.pl

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org/bovine240.htm) with 6.1-fold genome cover-
age by using radioactively labeled DNA probes
specific for those genes. Nineteen BAC clones
were identified by hybridization with mixed
CSN1S1- and CSN3-specific probes (Table 1).
The
CSN1S1-,
CSN1S2-,
and CSN3-specific primers (Table 1) were used to
confirm the presence of the sequences of interest
in those BAC clones. Eight BAC clones were de-
CSN2-,
STATH-
tected as positive for at least one primer pair
(Table 2).
TheCH240-130F23
clones were confirmed to contain CSN1S1,
andCH240-035O12
CSN1S2, CSN2, STATH and CSN3 genes (Ta-
ble 2). In the casein gene cluster, the distance be-
tween bovine CSN1S1 and CSN3 is about 250 kb
(Rijnkels et al. 2003). The insert size of BAC
clonesfrom bovine genomicBAClibrary
244A.A. Sazanov et al.
Table1.PrimersusedforconfirmationofthepresenceofthesequencesofinterestintheCHORI-240BACclones
Locus Gene Bank IDPrimersAnnealing temperature
(°C)
PCR fragment size
(bp)
CSN1S1
X59856
fw 5'-ATCCAAAGGCTTCAACTGCT-3'
rv 5'-CCTTTGACTCAGTGGCCTTT-3'
59 178
CSN1S2
M94327
fw 5'-TCTCCTCCTAGGATTGGAAAGA-3'
rv 5'-TTGAGACGGTTTGGGAATCT-3'
59 199
CSN2
X14711
fw 5'-ACAGCCTCCCACAAAACATC-3'
rv 5'- AGACTGGAGCAGAGGCAGAG -3'
60 224
STATH
AY154893
fw 5'-TGAAGAGGAACACCGGCTTA-3'
rv 5'-ATACAGCAAGAGGGCAGGAA-3'
60 212
CSN3
AY380229
fw 5'- ATTTATGGCCATTCCACCAA -3'
rv 5'- GATCTCAGGTGGGCTCTCAA-3'
60 166
Table 2. Summary of PCR confirmation results
BAC clone
CSN1S1CSN2STATH CSN1S2 CSN3
CH240-046B08
––––+
CH240-035O12
+++++
CH240-115O19
–++++
CH240-130F23
+++++
CH240-124F17
–––––
CH240-126C05
–––––
CH240-258E02
–––––
CH240-200N21
–++++
CH240-254P04
–––––
CH240-260O08
–––––
CH240-274P22
–++++
CH240-270N05
––––
CH240-250M04
–++++
CH240-262B19
–––––
CH240-200L09
–––––
CH240-228M10
–++++
CH240-233L10
–––––
CH240-215M02
–––––
CH240-205K17
–––––

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CHORI-240 Segment I is on average 180 kb,
and ranges from <100 kb to 270 kb (http://bacpac.
chori.org/bovine240.htm). Insert sizes were eval-
uated by cutting of CH240-130F23 and CH240-
035O12 clones with restriction enzyme NotI. The
insert sizes were 265 kb and 263 kb in
CH240-130F23 and CH240-035O12 clones, re-
spectively. So, the CH240-130F23 and CH240-
035O12 clones include all the coding sequences
of the cluster and, probably, up to 20 kb of the 5’
flankingregion. Itseemsverypromisingtosearch
for putative LCRs (locus controlling regions)
within the clone CH240-130F23, because such el-
ements are usually located in 5’ flanking regions
up to –35 kb upstream of coding genes (Li et al.
1999). Five clones were shown to give positive
PCR results with CSN2, STATH, CSN1S2 and
CSN3 genes (Table 2). These data showed that the
BAC contig was established for the whole casein
cluster, including all the 5 known genes (Table 2;
Figure 1). In the Bovine Genome Database
(http://www.ncbi.nlm.nih.gov/genome/guide/cow/in-
dex.html) the STATH and CSN1S2 are located in the
NW_936323 contig at
and 26 59538 541, respectively. The CSN2
and CSN1S1are in the contig NW_973412 at posi-
tions 418 544426 774 and 445 522463 059, respec-
tively. The CSN3 gene is not referred to in the
Bovine Genome Database. So, we can conclude
that the distance between STATH and CSN1S2
positions8211090
is 25 505 bp, and between CSN2 and CSN1S1
is 18 748 bp. Basing on the known insert sizes
of BAC clones in the CHORI-240 library and esti-
mated distance in approximately 200 kb between
CSN1S1 and CSN3 (Rijnkels et al. 2003), we con-
clude that this contig could contain up to 70 kb
of flanking regions. A maximum 7-fold coverage
of the contig is assumed in the 3’ region of the
clusterincluding
CSN2,
and CSN3 genes.
STATH,CSN1S2
Acknowledgements. This work was supported by
grants from the State Committee for Scientific Re-
search, Warsaw, Poland (KBN 3P06D00523) and
NATO (LST.CLG.979802).
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BAC clones containg bovine casein genes245
Figure 1. The CHORI-240 BAC contig containing the bovine CSN1S1, CSN2, STATH, CSN1S2 and CSN3 genes