Journal of Andrology, Vol. 24, No. 6, November/December 2003
Copyright © American Society of Andrology
Abundance of Repetitive Sequence Elements in the Mouse Testis-Specific Lactate Dehydrogenase-C Gene1
PER G. OLSSON*,
HIROSHI TSUJIOKA
,
SONOKO NARISAWA,
ERWIN GOLDBERG
AND
JOSÉ LUIS MILLÁN*,
From the * Department of Medical Biosciences,
Medical Genetics, Umeå University, Umeå, Sweden;
The Burnham Institute, La Jolla, California;
and the Department of Biochemistry, Molecular
Biology and Cell Biology, Northwestern University, Evanston, Illinois.
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Correspondence to: Dr José Luis Millán, The Burnham Institute,
10901 N Torrey Pines Rd, La Jolla, CA 92037 (e-mail:
millan{at}burnham.org). |
| Received for publication April 21, 2003;
accepted for publication July 21, 2003. |
 |
Abstract
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We have cloned and sequenced the entire mouse ldhc gene and mapped
it physically in relation to the somatic ldha gene. The 2 genes were
found to be oriented in head-to-tail fashion with about a 6-kilobase (kb)
distance between the 3' end of ldha and the 5' end of
ldhc. The ldhc gene is composed of 43% repetitive elements
compared to only 16% in the ldha gene. Despite the close physical
distance of mouse ldha and ldhc, the 2 genes have a very
different content of repetitive elements, and this most likely reflects
different levels of selective pressure.
Key words: Isozyme, restriction maps
Lactate dehydrogenase (LDH)-C is the LDH isozyme found in the germinal
epithelium during spermatogenesis and is most likely adapted to satisfy the
metabolic requirements of the differentiating germ cells and functional
spermatozoa (Blanco, 1980). It
is unclear, however, if LDH-C is essential for spermatogenesis or if other
isozymes (LDH-A and LDH-B) would perform the same function(s) in the absence
of LDH-C. One way of acquiring insight would be to create animals that lack
LDH-C by a targeted disruption of the ldhc gene. However, a number of
attempts to create a "knockout" of the mouse ldhc gene
have failed. In order to try to understand the reason for these failures, we
cloned and sequenced the entire mouse ldhc gene.
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Materials and Methods
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The RPZD mouse p1-derived artificial chromosome (PAC) library was obtained
from the Resource Center of the German Human Genome Project (RZPD; Berlin,
Germany) and screened using the entire mouse ldhc complementary DNA
(Wu et al, 1987) as a probe,
and 2 confirmed positive clones (RPCIP711P15209Q3 and RPCIP711P01257Q3, here
referred to as 209 and 257) were analyzed further. PAC DNA was isolated using
a standard protocol (Meier-Ewert et al,
1998), and restriction maps of the parts containing mouse
ldhc were made. Because the restriction patterns of the 2 PACs
differed markedly, the exon content of the 2 PAC clones was investigated using
polymerase chain reaction (PCR)-generated exon-specific probes. This showed
that PAC 209 contained exons 1-5 while PAC 257 contained exons 5-8 and that
the overlap between the 2 clones was about 1 kilobase (kb)
(Figure, A). The PACs were
subcloned into pBluescript (Stratagene, La Jolla, Calif) using the restriction
enzymes BamHI, PstI, XbaI, and KpnI. The
subclones were screened for their exon content and sequenced by dye terminator
cycle sequencing from the ends using exon-specific primers. Where needed,
primer walking generated additional sequencing data. Sequences were then
assembled using Assemblylign software. Mouse ldhc and mouse
ldha genes have previously been mapped close to each other on mouse
chromosome 7 (Stubbs et al,
1994). As it was possible that one of the two PACs that covered
the mouse ldhc gene also contained parts of the mouse ldha
gene, we used 3 sets of primers specific for mouse ldha (3',
exon4, and 5') to determine the relative orientation and distance of the
2 genes. PAC 209 was positive for ldha in all 3 PCRs, while PAC 257
was negative, placing mouse ldha 5' of mouse ldhc.
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(A) Map of the ldhc locus. Thicker black line denotes the
sequenced areas; boxes denote exons (open when untranslated), ldha in
blue and ldhc in green. Restriction enzymes: E = EcoRI, K = KpnI, N =
NcoI, and X = XbaI. Primers used for mapping included the following:
ldhc exon2: 5'-ATA AACTTTCCCGGTGTAAG-3' and
5'-ATACTAATAGCACACGCCATG-3', ldhc exon3:
5'-GGTTTGGCTGATGAACTTGC-3' and 5'-CAAAGACGATTT
TTGGAGTGC-3', ldhc exon4:
5'-ATCTGCCAACTCCAAACTGG-3' and
5'-ACTGGGTTAGTGACGATAAT-3', ldhc exon5:
5'-TTGACATACGTGGTT TGGAAG-3' and
5'-CCATGTTCTCCAAGAACCCA-3', ldhc exon6:
5'-TGCCCATATGGAGTGGTGTA-3' and
5'-CACCACCTGCTTGTGAACAT-3', ldhc exon7:
5'-CGGCTATGAGGTCCTTAACA-3' and
5'-CTTAACCAGCGTGGTAACAG-3', ldhc exon8:
5'-GGCTTCCATGGGATAAAGGA-3' and 5'-CTGTTA
CCACGCTGGTTAAG-3', ldha 5':
5'-TTTCTTTGGGGTGTCGCAG-3' and
5'-GCCTTAAATGGAAGCTCCG-3', ldha exon4:
5'-CAAGCTGGTCATTAT CACCG-3' and
5'-GGATTGGAGACGATCAGCAG-3', and ldha 3':
5'-TCTCGGATGTTGTGAAGGTG-3' and
5'-TTTCCCCACACCATCTCAAC-3'. (B) Genomic organization and
location of repetitive elements in the ldha gene, derived from the sequence
available in GenBank, and of the ldhc gene as reported in this paper. Orange
boxes denote SINE (short interspersed nuclear element) repeat elements (B1,
B1F, B2, B3, B3A, MUSID2, MUSID4, RSINE1, RSINE2, and RSINE2A), red boxes
denote LINE (long interspersed nuclear element), transposon, and endogenous
retrovirus elements (L1MB6_5, L1P_MA2, MERVL, RMER6A, and URR1), and purple
boxes denote short tandem repeats (ie, (GGGGA)n, (CAAAA)n, (CA)n, and
(A)n)).
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Results
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Computer-generated restriction maps of the sequences of the ldha
and ldhc genes were compatible with a head-to-tail orientation of the
2 genes, and this was confirmed by Southern blot analysis using the mouse
ldha 3' PCR product and a 134-bp SphI-SacI
fragment 5' of mouse ldhc as probes
(Figure, A). As predicted, both
probes shared the same size of NcoI, KpnI, and XbaI
fragments but had different EcoRI fragments, and double-digest using
EcoRI in combination with NcoI and XbaI confirmed
the predicted map. Thus, the end of the mouse ldha exon 8 is about 6
kb from the 5' beginning of the mouse ldhc exon 1
(Figure, A). The entire mouse
ldhc, including some flanking regions, was sequenced, and the
intron-exon boundaries were identified. The total distance between mouse
ldhc exons 1 and 8 is about 16 kb, and introns 2, 3, and 4 are all
relatively large (3-4 kb in length), while introns 1 and 6 are shorter (<1
kb) (Figure, A).
Since previous attempts to generate knockout mice appear to have failed
because of a high proportion of repetitive sequences in constructs containing
the 5' end of the gene, the PAC clones were initially screened for
repetitive elements using mouse Cot-1 (Invitrogen Corp, Carlsbad, Calif)
hybridization. Surprisingly, most of the Cot-1-positive bands were the same as
the ones positive for mouse ldhc, indicating that while much of the
area surrounding the gene is relatively free from repeats, the mouse
ldhc gene appears to contain a large number of repetitive elements.
We compared the 20-kb ldhc gene sequence to the "Censor"
(Jurka et al, 1996) repeat
database, and the results indicated that, indeed, 43% of the gene sequence was
composed of repetitive elements. Only a 1.3-kb area around exon 4 and the
entire intron 7 were free of repetitive elements. The adjacent mouse
ldha gene was more compact (10 kb between exons 1 and 8)
(Fukasawa and Li, 1987) than
the mouse ldhc gene, and, when analyzed for repetitive elements, its
content was significantly lower—16%. This is in agreement with our Cot-1
hybridization result, which indicated that the region adjacent to the mouse
ldhc gene contained fewer repetitive elements than the gene itself
(Figure, B).
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Discussion
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The 3 LDH isozymes from the mouse (and other mammals) share about 70% amino
acid homology with each other. LDH-A and LDH-B have been conserved stringently
during evolution; LDH-C has diverged more between species. While the mammalian
LDH-B subunits show a conservation of the amino acid identity around 95% and
the LDH-A subunits show 90%, the LDH-C homology drops to 75% conservation
between mammalian species, which means that LDH-C proteins are as similar to
each other as to the other 2 isozymes. Even at larger evolutionary distances,
such as those that exist between the mouse and Xenopus laevis, the
LDH-A and LDH-B proteins are more than 80% identical, which is higher than the
identity between mouse and human LDH-C—74%. This means that the
ldhc gene is allowed to diverge at a much higher rate than
ldha/ldhb and must thus be subject to a different and less stringent
selective pressure.
The tandem orientation of ldha and ldhc genes is also
found in humans. The evolution of the ldh genes in vertebrates
through fishes indicates that they stem from a common ancestor that was
duplicated before the speciation of mammals. Indeed, the evolutionary tree
suggested by Tsuji et al (1994)
indicates that the duplication of ldha and ldhc occurred
before ldhb existed. On the other hand, Markert et al
(1975) proposed that the
duplication of ldha gave rise to ldhb and that, in fishes,
it was the duplication of the b gene that led to the appearance of
ldhc in the eye or liver, depending on the species. We suggested
(Millán et al, 1987)
that the orientation of ldha and ldhc genes and the relative
homology of the coding regions of the 3 LDH subunits indicate that
ldhc represents a duplication of ldha. Despite the close
physical distance of mouse ldha and ldhc, the 2 genes have a
very different content of repetitive elements, and this most likely reflects
the different levels of selection under which they have operated. The
accumulation of repetitive elements in the mouse ldhc introns has
doubled the size of the gene compared to ldha. The reason why the 2
genes have been kept in close proximity is unclear; it may be pure chance, but
there may also be functional constraints that keep the 2 genes together,
possibly common regulatory elements situated between the 2 genes.
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Acknowledgments
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This work was supported in part by grant HD05863 from the National
Institutes of Health and a grant from the Swedish Medical Research
Council.
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Footnotes
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1 GenBank accession number AF190799. 
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