Biochimica et Biophysica Acta 1579 (2002) 203 – 206
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Short sequence-paper
Sequence of goat cyclin T1 cDNA, gene organisation
and expression analysis
Xavier Mata *, Sead Taourit, Jean-Luc Vilotte
Laboratoire de Génétique Biochimique et de Cytogénétique, INRA, 78352 Jouy-en-Josas Cedex, France
Received 26 July 2002; received in revised form 20 September 2002; accepted 25 September 2002
Abstract
The cyclin T1 (Cyc T1) protein has been recently identified, associated with the cyclin-dependent kinase 9 (CDK 9), as to be involved in
the transcriptional activation of the Human Immunodeficiency Virus type 1 (HIV-1) by the Tat protein. In this study, the sequence of the 7 kb
goat Cyc T1 cDNA is reported as well as the exon/intron structure of the gene. Its observed ubiquitous expression is consistent with the
promoter structure.
D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Cyclin T1; HIV-1; cDNA; Gene structure; Promoter analysis; Goat
The cyclin T1 (Cyc T1) protein was recently identified as
being a co-factor for the Human Immunodeficiency Virus
type 1 (HIV-1) Tat Protein, in association with the cyclindependent kinase 9 (CDK 9) [1,2]. This complex is involved
in the RNA pol II transcription elongation. The interaction
of the Tat protein with the Cyc T1/CDK 9 complex strongly
increases the affinity and specificity of the Tat protein for
the cis-acting transcription response element (TAR), making
a stable RNA stem-loop structure at the 5Vend of nascent
viral transcripts [1,2].
Transcription of the human Cyc T1-encoding gene results
in the synthesis of an 8-kb-long transcript in most tissues
analysed. The gene appears to be ubiquitously expressed and
is located on the human chromosome region 12 pter-qter.
Structural analysis of the mRNA is still incomplete. So far,
only its 5V-UTR and open reading frame (ORF) sequences
have been reported in human, mouse and horse species [1– 4]
(AF109179, AF190905). In the three abovementioned species, it only covers around 2 kb of the cDNA including only a
few nucleotides of the 3VUTR. Nevertheless, by comparison
with the human genome sequence, the partial structure of the
corresponding gene has been determined and is reported in
GenBank NT 009526. The sequence of the promoter region
* Corresponding author. Tel.: +33-1-34-65-25-76; fax: +33-1-34-6524-78.
E-mail address: [email protected] (X. Mata).
has also been determined for the human and murine genes [6]
and characterised both by sequence similarity analyses and
transfections in various cell types [5,6].
We have recently reported the site-independent expression
of a goat a-lactalbumin BAC transgene in mice [7] and the
subsequent discovery within this BAC of the Cyc T1 gene
and of its ubiquitous expression in transgenic mice [8]. To
further define this chromosomal region, we have investigated
the genomic structure of the Cyc T1 gene. In the present
paper, we report the full-length cloning and sequence analysis
of the goat Cyc T1 cDNA, the deduced intronic/exonic
organisation of the corresponding gene and the determination
of its proximal promoter sequence. Expression of this gene
was studied by Northern blot analysis and RT-PCR in various
goat tissues and was confirmed to be ubiquitous also in
ruminants.
The cloning of the full-length goat Cyc T1 cDNA has been
realised in several steps. From a precedent work (Vilotte,
unpublished data), involving partial sequencing of the goat alactalbumin BAC insert, two exons of the Cyc T1-encoding
gene were identified (exons 7 and 8, Fig. 1B). This sequence
information allowed us to define two primers (A and B in
Table 1) that were used to perform a 5VRACE RT-PCR
reaction using 6 Ag of total RNA from kidney and lactating
mammary gland, following the manufacturer’s instructions
(SuperScript First-Strand Synthesis System for RT-PCR,
Invitrogen). With both RNA samples, a fragment of 830 bp
was amplified, cloned and sequenced. It contained 72 bp of
0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 7 - 4 7 8 1 ( 0 2 ) 0 0 5 4 2 - 0
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X. Mata et al. / Biochimica et Biophysica Acta 1579 (2002) 203–206
Fig. 1. Schematic representation of the Cyc T1 genomic DNA. (A) Representation of the BAC insert. Grey arrow: Cyc T1 gene. Black arrow: a-lactalbumin
gene. Both loci are in the same orientation, as indicated by the arrows. (B) Structural organisation of the Cyc T1 transcription unit. Black boxes: coding exons.
Grey boxes: 5Vand 3VUTR (72 and 4554 bp, respectively). Both representations are not at scale. Sizes of introns and exons are reported in Table 2.
5VUTR and part of the ORF of the cDNA. To ensure that the
cloned 5VUTR was complete, this same experiment was
repeated using primers located further upstream (C and D
in Table 1). All amplified fragments ended at the same
nucleotide (nt), strongly suggesting that a unique transcription start site is present within the goat Cyc T1 gene (data not
shown). This is in contrast with the human gene which has
several potential transcription initiation sites, at least as
determined in vitro [5].
The abovementioned 830 bp partial cDNA was used as
probe to screen a goat ovarian cDNA library, a kind gift from
Dr. Pailhoux (INRA, France). 1.75 106 pfu of the unamplified oligo-dT primer library were plated and screened. Two
positive clones were identified. Sequence analysis of their
inserts revealed that both contained the whole ORF and 460
bp of 3VUTR. Analysis of the sequence upstream of the 3Vend of both cDNAs did not reveal the presence of a polyadenylation signal sequence. Screening of three other cDNA
libraries from ovaries or testicular origins did not allow us to
isolate cDNA clones with longer 3VUTR sequences (data not
shown). Furthermore, Northern analysis performed on several goat tissues revealed a Cyc T1 mRNA size similar to the
Table 1
Name and position of the different primers used during this work
Primer name
Position on the cDNA
Orientation
A
B
C
D
E
F
G
H
747 – 765
728 – 746
290 – 310
263 – 284
6142 – 6161
6150 – 6169
6153 – 6173
6401 – 6523
Reverse
Reverse
Reverse
Reverse
Forward
Forward
Reverse
Forward
Positions refer to GenBank AF506739 sequence.
human mRNA [1] (see below). Both observations strongly
suggested that the cloned cDNAs resulted from retro-transcription events initiated within the mRNA sequence rather
than at its 3V-end.
Attempts to clone the missing 3VUTR by 3VRACE RTPCR were unsuccessful. In order to obtain sequence information on it, we aligned 15 kb of the human sequence
(NT 009526) located downstream of the Cyc T1 stop codon
with the human EST database. It revealed that the 5 kb
downstream of the stop codon were homologous to a set of
overlapping EST (data not shown). The most 3V-located of
these ESTs (GenBank AI052701) contained a polyadenylation signal, suggesting that it derived from the 3V-end of a
mRNA. Altogether, these observations were consistent with
the hypothesis that the 3V-UTR of the Cyc T1 cDNA is
comprised within a single exon encompassing the end of
the ORF. Analysis of Ruminant ESTs revealed that a bovine
EST (GenBank AW345578) was homologous to the human
sequence 4 kb downstream of the Cyc T1 stop codon, thus
within the putative 3VUTR of the gene. We used this EST to
design three primers (E, F and G in Table 1) to clone the
3VUTR region by RT-PCR and 3V-RACE RT-PCR, using the
corresponding kits from Invitrogen, 6 Ag of goat kidney total
RNA and following the manufacturer’s instructions. The RTPCR experiment allowed us to clone the cDNA region
located between the stop codon and nt 6191 in GenBank
AF506739. The 3V-RACE RT-PCR strategy resulted in the
isolation of a 280-bp-long fragment whose 3V-end corresponds to nt 6430 in GenBank AF506739. It was located
upstream to the hypothesised 3Vend of the human gene
transcription unit. Furthermore, no polyadenylation signal
was observed within its sequence. It strongly suggested again
that this amplified cDNA resulted from a retro-transcription
event initiated within the mRNA sequence. To confirm it, a
X. Mata et al. / Biochimica et Biophysica Acta 1579 (2002) 203–206
Table 2
Size of the goat and human exons as well as estimation of the goat and
human introns length
Size
1
2
3
4
5
6 7
8
9
Goat exons
(bp)
Human exons
(bp)
Goat introns
(kbp)
Human introns
(kbp)
232
82
127
60
62
45 163
70
5937
169 – 412 81
128
60
62
45 163
70
?
3.8
2
14
8.5
2.5 1.3 1.3 1
0.165 10
4.7 1.2 1.6 2
0.168 1.3
new 3VRACE RT-PCR experiment was performed using
oligonucleotide H in Table 1. It resulted in the amplification
of a 406-bp-long cDNA whose 3V-end was this time identical
to that suspected in the human sequence. A polyadenylation
signal was present 15 nt 5Vof the polyadenylation site.
Sequence analysis confirmed the occurrence of an A-rich
region around nt 6430, indirectly reinforcing our argument
for an artefactual initiation of the reverse-transcription in the
previous 3V-RACE RT-PCR experiment.
The 72 bp 5VUTR of the cDNA has been well conserved
during evolution as judged by the high homology observed
between the horse, human, mouse and goat sequences,
which is at least of 88%. This suggests that this region
has a regulatory function, acting either at a co- or posttranscriptional level.
The ORF of the Cyc T1 is also well conserved between
these species with 92% identity with the horse, 90% with the
human and 86% with the mouse sequences. All amino acid
motifs described as having important functions are conserved
in the goat species. This includes the cyclin box (aa 1 – 250),
the potential Tat/TAR recognition motif (aa 254 –272), a
putative coiled coil domain (aa 379– 430), the poly-HIS (aa
517 –528) and the poly-SER (aa 560 –570) [1] as well as a
carboxy-terminal PEST sequence that are commonly found in
G1 cyclins and serve to regulate protein turnover by cellular
ubiquitination and proteolysis pathways [9]. Mouse cells do
not sustain HIV replication. This observation was attributed
to one amino acid polymorphism that differentiate the human
and the murine Cyc T1 protein [2]. Occurrence of a tyrosine at
position 261 in the mouse protein in place of a cysteine
confers the loss of HIV-1 Tat transactivation. In the goat
sequence, a tryptophane residue is found at this location. To
our knowledge, no HIV-like illness has ever been described in
goats. Whether or not this data is related to the occurrence of a
tryptophane at position 261 of the Cyc T1 protein is an
attractive hypothesis that remains to be tested.
Notably, a very long 3VUTR (4.5 kbp) has been found in
the goat Cyc T1 mRNA. The estimated sizes of the human
Cyc T1 mRNAs suggest that this long 3VUTR is a common
feature [1]. Interestingly, this long sequence was found to be
well conserved between the human and the goat genes, with
81% homology, reinforcing the hypothesis that it has an
important function. It was proposed that this region might
205
be involved in the post-transcriptional regulation of the
expression of this gene [5], through a translational control
mechanism. Several A/T-rich regions are present, and conserved in this 3VUTR. These regions are known to be the
target of endonucleases that regulate the stability of a variety
of transcripts. Thus, beside playing a potential role in the
translational control of the mRNA, the 3VUTR may also be
involved in stability control mechanisms.
Alignment of the partial BAC sequence with the Cyc T1
cDNA was performed to determine the structure of the goat
Cyc T1 gene transcription unit (Fig. 1B, GenBank
AF506740-506747). Nine exons, whose sizes vary between
45 bp and 6 kbp, were identified (Fig. 1B and Table 2). When
necessary, estimation of the length of the introns was performed using the long-range PCR kit (Expand Long Template
PCR System, Roche) (Table 2). The complete transcription
unit of the gene spans almost 42 kb and is thus longer than
that observed in human. This results mostly from the longer
sizes of introns 2 and 8. Differences in the size of introns are
often associated with insertion and/or deletion of repetitive
elements (not determined). All sequenced intron boundaries
follow the ‘‘exon n-GT-intron n-AG-exon n + 1’’ rule [10,11].
Within the goat BAC, the Cyc T1 gene was found to be
located 80 kb apart from the a-lactalbumin locus (Fig. 1A)
[12]. The orientation of the transcription unit of the two genes
was the same (Fig. 1A, data not shown).
Alongside the exon 1, 2005 bp of the 5V-flanking region
were cloned and sequenced. Functional analysis of the
homologous human promoter in cell cultures suggested that
its activity resides in the 545 nucleotides upstream to the
translation initiation site [5,6]. Furthermore, several potential binding sites for transcription factors were identified
within this proximal promoter and found to be conserved in
the murine sequence [6]. Alignment of the goat sequence
revealed that some of these putative regulatory elements are
conserved in the three species, including consensus binding
Fig. 2. Northern analysis of the goat Cyc T1 gene expression. Northern blot
was performed with 1.4 Ag of goat mammary gland (MG) and 3.5 Ag of
goat skeletal muscle (Mu) polyA+ RNA. Hybridisation with an oligolabelled Cyc T1 cDNA was performed as already described [5]. Sizes of the
hybridising mRNAs were estimated using 18S and 28S distance of
migration as well as a co-migrated 1-kb ladder size marker and are
indicated.
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X. Mata et al. / Biochimica et Biophysica Acta 1579 (2002) 203–206
sites for Sp1 and members of the Ets family of transcription
factors (data not shown). This conservation across evolution
could suggest that these sequences are indeed involved in
the transcriptional regulation of the Cyc T1 promoter,
although direct evidence of it is lacking. The homology
between the 5V flanking region of this gene, with that of the
two different species mentioned above, remains significant
only over a short length of about 0.5 kb, indirectly reinforcing the hypothesis that this sequence contains most if not all
of the promoter activities [5].
The human and murine Cyc T1 promoters are known to
have an ubiquitous expression [6]. RT-PCR and Northern
analyses were performed using goat RNA from five different tissues: mammary gland, testis, ovaries, kidney and
skeletal muscle. RT-PCR showed that the goat gene is also
ubiquitously expressed (data not shown), confirming the
expression pattern obtained with this gene in transgenic
mice [8]. The low expression level of this gene complicated
its analysis by Northern blotting. Nevertheless, it revealed
that the transcript that was detected in all tissues was of
approximately 7 kb in length, a result concordant with the
size of the cDNA (Fig. 2). Surprisingly, a shorter transcript
was also detected in the skeletal muscle (Fig. 2). This
transcript was of 1.5 –2 kb in length. Origin and biological
significance of this mRNA remain unknown. The estimated
size of this transcript is such that it cannot encompass the
entire Cyc T1 ORF and is too short to correspond to the
‘‘partial’’ cDNAs cloned from the abovementioned libraries.
Southern blotting hybridisation of PstI or EcoRI digested
goat genomic DNA suggested that the Cyc T1 gene is a
single copy gene (data not shown), consistent with the
single FISH signal observed with this gene [7]. Thus, this
transcript must derive from unusual splicing and/or termination events of the Cyc T1 gene. A shorter Cyc T1
transcript has been observed in some human tissues, the
origin of which was not described [1]. However, its estimated size of 3 kb is much longer that the one determined
for the short transcript present in the goat skeletal muscle.
The structure of the goat Cyc T1 promoter is consistent
with its pattern of expression. This promoter lacks TATA
and CAAT boxes and contains several potential ubiquitous
transcription factor binding sites located close to the transcriptional start site (like Sp1, Ets family, AP1. . .). It is also
characterised by a relatively high G/C content (55 – 60%).
These characteristics of housekeeping gene promoters are
also shared by its human and murine counterparts and by
several cycle-regulated promoter genes [13]. Ubiquitous
promoters are potentially interesting for building gene
therapy vectors. The previously described ubiquitous
expression of the goat Cyc T1 gene in transgenic mice [8]
and the confirmation of the potentially compact organisation
of its promoter, as suggested by the currently reported
sequence analysis, make it a good candidate among other
house keeping genes. Its ability to target expression of
reporter genes in currently tested in mice.
Acknowledgements
We are most grateful to Dr. Pailhoux (INRA) for the kind
gift of cDNA libraries and goat RNA samples, to Dr. Persuy
(INRA) for providing mammary gland mRNA and to Dr. Le
Provost (INRA) for critical reading of this manuscript and
mRNA samples.
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