ABSTRACT
A truncated cDNA clone encoding
Tetrahymena
thermophila
histone H2A2 was isolated using synthetic degenerate oligonucleotide probes
derived from H2A protein sequences of
Tetrahymena
pyriformis
. The cDNA clone was used as a homologous probe to isolate a truncated genomic
clone encoding H2A1. The remaining regions of the genes for H2A1 (
HTA1
) and H2A2 (
HTA2
) were then isolated using inverse PCR on circularized genomic DNA fragments.
These partial clones were assembled into intact
HTA1
and
HTA2
clones. Nucleotide sequences of the two genes were highly homologous within the
coding region but not in the noncoding regions. Comparison of the deduced amino
acid sequences with protein sequences of
T.pyriformis
H2As showed only two and three differences respectively, in a total of 137
amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes
arose before the divergence of these two species. The
HTA2
gene contains a TAA triplet within the coding region, encoding a glutamine
residue. In contrast with the
T.thermophila
HHO
and
HTA3
genes, no introns were identified within the two genes. The 5
'
- and 3
'
-ends of the histone H2A mRNAs were determined by RNase protection and by
PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly
expressed in vegetatively growing cells but only weakly expressed in starved
cultures. With the inclusion of these two genes,
T.thermophila
is the first organism whose entire complement of known core and linker
histones, including replication-dependent and basal variants, has been cloned and sequenced.
The DNA of eukaryotes is packaged into nucleosomes consisting of a core and a
linker region. The core is composed of two molecules each of histones H2A, H2B,
H3 and H4, around which is wound ~146 bp of DNA in 1
3
/
4
left-handed superhelical turns. The linker consists of variable amounts of DNA
associated with one molecule of histone H1 (reviewed in
1
). The histones are basic proteins which have been highly conserved in
evolution, albeit to somewhat different degrees (H4=H3 > H2B=H2A > > H1; see
1
,
2
).
In most eukaryotes, each histone class is encoded by a small multigene family.
Expression of most major histone genes is coupled to DNA replication. In
contrast with these replication variants, some histone genes encode
replacement/basal variants that are transcribed throughout the cell cycle and
in non-growing cells. The yeast
Saccharomyces cerevisiae
is the only organism in which all of the genes for all of the known histones
have been cloned and sequenced and in which the function of histone genes
modified
in vitro
can be studied
in vivo
using transformation and gene replacement. Unfortunately, yeast is unusual
among eukar- yotes in lacking histone H1 and distinct replacement variants of the core
histones (
3
-
5
).
The histone complement of the ciliated protozoan
Tetrahymena thermophila
has been extensively studied and shown to contain both histone H1 and
replacement variants (reviewed in
2
,
6
,
7
). Recently, methods for mass transformation (
8
) and for gene replacement (
9
-
11
) have been developed for
Tetrahymena
that should enable detailed functional analyses of histone genes in that
organism, including analyses of H1 and of minor replacement variants that are
lacking in yeast. A prerequisite to such an analysis is the cloning of the
histone gene complement of
Tetrahymena
. To date, genes encoding all of the known
Tetrahymena
histones except for two major H2As have been cloned and sequenced (
2
,
12
-
20
). In this report we describe the structure, sequence and expression of the
T.thermophila HTA1
and
HTA2
genes encoding histone H2A1 and H2A2 and summarize the organization of the
first complete histone gene complement from an organism containing all of the
histone subtypes common to most eukaryotes.
Tetrahymena
thermophila
(strain CU 428, mating type VII) were grown and harvested, nuclei isolated, and
DNA and RNA prepared as previously described (
16
,
21
-
23
).
Restriction digests and Southern blots were carried out according to standard
protocols (
24
).
Based on codon usage information available for
Tetrahymena
histone genes (
15
,
25
and T. Thatcher and M. A. Gorovsky, unpublished observations) two degenerate
oligonucleotides were synthesized corresponding to two regions (amino acid
residues 73-80 and 90-96) that are highly conserved in all H2As including those of
Tetrahymena
pyriformis
(
26
).
Probe 1 G C {T over C} G C {T over C} A A {A over G} G A {T over C} A A {T over G} A A
{A over G} A A {A over G} A C N
{A sub {{size 8 {7 3}}}} {A sub {{size 8 {7 4}}}} {K sub {{size 8 {7 5}}}} {D
sub {{size 8 {7 6}}}} {N sub {{size 8 {7 7}}}} {K sub {{size 8 {7 8}}}} {K sub
{{size 8 {7 9}}}} {T sub {{size 8 {8 0}}}}
Probe 2G C {T over C} A T {T over C} A G A {T over C} A A {T over C} G A {T over C} G A
{A over G} G A {A over G}{A sub {{size 8 {9 0}}}} {I sub {{size 8 {9 1}}}} {R sub {{size 8 {9 2}}}} {N
sub {{size 8 {9 3}}}} {D sub {{size 8 {9 4}}}} {E sub {{size 8 {9 5}}}} {E sub
{{size 8 {9 6}}}}
Probes were labeled with [[gamma]-
32
P]ATP and polynucleotide kinase (
24
) to a specific activity of >1 * 10
8
c.p.m./[mu]g. The optimum conditions for both probes were hybridization at 30oC, followed by washing at 35-40oC. Hybridization solution was 5* SSPE, 1* SPED (8* SPED = 0.8% Ficoll, 0.8% PVP360, 0.8%
BSA, 48 mM SDS, 16 mM pyrophosphate, 16 mM EDTA). Washing was done in 2* SSPE and 0.1% SDS.
A library of
T.thermophila
log phase cDNAs in [lambda]gt11 constructed by D. Shapiro (
16
) was probed with the two oligonucleotides under the conditions described above.
Plaques that hybridized positively to both probes were selected and re-screened. Phage DNA was isolated essentially as described by Maniatis
et al
. (
27
) with the following modifications. Phage lysate was spun twice to remove
contaminating chromosomal DNA. Fifty [mu]l of 1 mg/ml DNase I and 25 [mu]l of 10 mg/ml RNase A were then added to 50 ml of supernatant and
incubated for 1 h at 37oC. After addition of 2.92 g of NaCl, 5 g of PEG and incubation on ice for
30 min, phage were pelleted at 12 000
g
for 10 min at 4oC, and solubilized in 2 ml of a solution containing 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.2% Sarkosyl. After addition of 5 [mu]l of 20 mg/ml Proteinase K and incubation at 55oC for 20 min, phage DNA was isolated by phenol-chloroform extraction and ethanol precipitation.
Although the library was constructed by cloning into the
Eco
RI site of [lambda]gt11, the cDNA inserts could not be released with
Eco
RI (subsequently, sequence information indicated that the junctions were
disrupted). Therefore,
Sac
I/
Kpn
I-digested insert fragments, which also include 2.1 kb of [lambda]gt11 sequences, were subcloned into
Sac
I/
Kpn
I sites on the plasmid vector Bluescript KS(+) (Stratagene) and sequenced using
a pair of [lambda]gt11 sequencing primers (forward primer = GACTCCTGGAGCCCG; reverse
primer = GGTAGCGACCGGCGC) according to standard protocols for double-stranded DNA sequencing (
24
). A truncated
HTA2
cDNA clone (pXL40) was identified.
The 145 bp
Acc
I-
Bal
I fragment from pXL40 was blunt-end ligated into the
Sma
I site of pBluescript to obtain pXL41, from which the insert could be released
with
Bam
HI and
Pst
I to obtain a homologous probe to facilitate cloning the remainder of the
Tetrahymena
H2A genes. Genomic DNA was digested with
Bam
HI,
Pst
I,
Eco
RI,
Hin
dIII and
Kpn
I and probed with the 145 bp
HTA2
cDNA sequence (data not shown).
Hin
dIII-digested fragments of 4.1 and 8.0 kb in size were identified and used for
construction of a size-selected genomic library. Fifty [mu]g of genomic DNA was digested and run on a 0.5% agarose gel. DNA
fragments between 3.5-4.5 and 7-9 kb were eluted as described (
28
), then ligated into Bluescript KS(+) vector, transformed into DH5[alpha] cells and screened (
24
). This method yielded a 4.1 kb truncated
HTA1
clone (pXL50).
Since both
HTA1
and
HTA2
clones obtained were truncated, we cloned the missing parts of each gene by an
inverse PCR method (
29
). When
Bgl
II-digested
Tetrahymena
genomic DNA was probed with the
HTA2
cDNA clone (pXL41), two bands, 2.2 and 2.0 kb in size, were observed.
Bgl
II-digested genomic DNA was ligated at a concentration of 2 [mu]g/ml to achieve 95% monomeric circles according to the formula of
Collins and Weissman (
30
). These ligated circles were then amplified with a single pair of primers,
Oligo 1408
CTTCAAGAATCTGG
derived from portions of the coding region of
HTA1
and
HTA2
known to be similar from sequencing the previously obtained partial clones.
Mutations (underlined) producing
Eco
RI sites were introduced into the two primer sequences to facilitate subsequent
cloning. PCR products were digested with
Eco
RI and cloned into the
Eco
RI site on the pKS(+) vector. Three independent colonies were obtained from
clones derived from each of two duplicate reactions, sequenced and compared to
detect any mutations occurring during PCR amplification. One completely correct
clone for
HTA1
(pXL51) and two correct clones for
HTA2
(pXL42a and pXL42b) were identified.
For construction of pXL53 containing the reconstituted
HTA1
gene encoding H2A1, the 1.9 kb
Bal
I-
Bgl
II fragment containing part of the coding sequences plus the entire 3' noncoding sequences from pXL51 was ligated into the
Bal
I-
Bam
HI sites of pXL50 with the removal of the small (159 bp)
Bal
I-
Bam
HI fragment from pXL50.
For assembly of pXL46 containing the intact
HTA2
gene encoding H2A2, pXL42b was cut with
Bgl
II and
Xba
I and resealed by blunt end ligation to remove the 3'-half of the gene as well as the
Xba
I site on the vector. The
Xba
I site within the region encoding H2A2 was not cut because this site is dam
s
. This gives pXL43. pXL40 and pXL43 were then grown in a dam
-
Escherichia coli
strain to isolate plasmids in which the
Xba
I sites could be cleaved. Then the 1.36 kb
Xba
I-
Sac
I (blunt) fragment of pXL40 was ligated into the
Xba
I-
Eco
RV sites on pXL43 to obtain pXL44. pXL42b was also digested with
Bgl
II and
Sal
I and blunt end ligated to remove the 5'-half of the
HTA2
gene, resulting in plasmid pXL45. The 1.66 kb
Bal
I-
Xho
I fragment from pXL45 was then ligated into the
Bal
I-
Xho
I sites of pXL44 with the corresponding fragment removed from pXL44. A schematic
presentation of the major steps of these procedures is given in Figure
1
.
For 5'-end analysis, PCR clones pXL51 (for
HTA1
) and pXL42a (for
HTA2
) were cut with
Bgl
II and transcribed with T7 RNA polymerase in the presence of [[alpha]-
32
P]UTP to generate probes with 105 nt of coding sequences plus 153 and 144 nt of
5'-flanking sequences for
HTA1
and
HTA2
respectively.
Two new constructs were made to map the 3'-ends of H2A messages. pXL52 (for
HTA1
) was obtained by ligating the 1.75 kb
Hin
dIII-
Bgl
II fragment of pXL51 into the
Hin
dIII/
Bam
HI sites of pBluescript KS(+) so that the antisense probe for the 3' region of
HTA1
messages can be generated by transcribing with T7 RNA polymerase after cleavage
with
Hin
dIII. pXL47 (for
HTA2
) was constructed by subcloning the 1.65 kb
Bal
I-
Bgl
II fragment of pXL42a into
Eco
RV/
Bam
HI-digested pBluescript KS(+). The antisense transcript for the 3' region of
HTA2
messages was synthesized by digesting with
Hin
dIII followed by T7 transcription.
RNase protection was done as described (
15
). Optimum RNase digestion conditions were found to be at room temperature for
15 min with RNase A at 8 [mu]g/ml and RNase T1 at 180 U/ml.
The 3'-ends of
HTA1
and
HTA2
messages were also mapped by the method of rapid amplification of cDNA ends
(RACE; see
31
-
33
). cDNA was made using an oligo(dT) adapter-primer (5'-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3') and then amplified using the adapter (the
above oligo without the 17 Ts) and Oligo 2407 (described above). PCR products
were cut with
Eco
RI (in Oligo 2704) and
Sal
I (in the adapter oligo), cloned into
Eco
RI/
Sal
I sites on pBluescript KS(+), and sequenced.
RNA ligase mediated RACE (RLM-RACE) was used to map both 5'- and 3'-ends of H2A1 and H2A2 messages as described (
23
).
Northern blotting of 1.2% formaldehyde-agarose gels was done according to standard protocols (
24
). The probe for
HTA1
and
HTA2
messages was derived from the 2 kb
HTA2
insert from pXL46, which recognizes both messages. The probe (phv1) for
HTA3
was derived from the genomic clone of
HTA3
(
17
) by the removal of the second exon (X. Liu and M. A. Gorovsky, unpublished
observations). Quantitation was done using a Molecular Dynamics PhosphorImagertm (Molecular Dynamics, Sunnyvale, CA).
When a [lambda]gt11 cDNA library (
16
) was screened with the two oligonucleotides based on amino acid residues 73-80 and 90-96 of histone H2A of
T
.
pyriformis
(see Materials and Methods), five positive clones were obtained which
hybridized to both probes. Inserts from these clones were purified, subcloned
into the plasmid vector Bluescript KS(+) and sequenced on both strands. When
the deduced protein sequences were compared with published
T.pyriformis
H2A protein sequences (
26
), they all were found to be truncated clones encoding H2A2 but missing the 3' half of the gene. The longest cDNA (pXL40) contained 26 bp of 5'-untranslated sequence and 354 bp of coding sequence,
encoding the first 117 of the 132 amino acid residues. We then constructed size-selected genomic libraries. When a
Hin
dIII-digested genomic Southern blot was probed with the
HTA2
cDNA clone, two bands, 4.1 and 8.0 kb, were observed (data not shown). We
succeeded in cloning only the lower band which contained a truncated
HTA1
clone (pXL50), containing ~3.7 kb 5' upstream sequence and 403 bp of coding sequence, encoding all
except the last four amino acid residues. Cloning of the upper band was
unsuccessful, probably because the AT-richness of
Tetrahymena
noncoding sequences makes cloning of large fragments extremely difficult (
34
).
Finally, inverse PCR (
29
) was used to clone the missing parts of the
HTA1
and
HTA2
genes. Two
Bgl
II fragments, 2.2 and 2.0 kb, respectively, were isolated corresponding to the
entire
HTA1
(pXL51) and
HTA2
(pXL42a, and pXL42b in the opposite orientation) clones except for the region
between the pair of primers used in the PCR reaction. This complex cloning
process is schematically presented in Figure
1
.
Since all of the clones contained incomplete genes, they were then assembled
into intact genes
in vitro
(Fig.
1
; for details see Materials and Methods). Briefly, the 3'-half of
HTA1
from the PCR clone (pXL51) was connected to the 5'-half of the genomic clone (pXL50) at the
Bal
I site in the coding region.
HTA2
assembly was somewhat more complicated. The final construct contained the 5'- and 3'-halves from the PCR clone (pXL42b) with the middle
part (
Xba
I-
Bal
I) from the cDNA clone (pXL40). The assembled constructs (pXL53 and pXL46 for
HTA1
and
HTA2
, respectively) now contain the full length genes. The coding regions from these
two clones were re-sequenced to check for any errors during the assembling procedures. None
were found. Also, these reconstructed genes have been shown to be able to
function as the only H2A genes in the yeast,
S.cerevisiae
(
5
). It is worth noting that, while this somewhat baroque scheme for cloning and
reconstructing the
HTA1
and
HTA2
genes is the most complicated one we have ever utilized for cloning any
Tetrahymena
gene, all of the more straightforward approaches we tried failed. It is also
worth noting that difficulties in cloning particular
Tetrahymena
genomic DNA fragments, especially larger ones, are not uncommon.
The DNA sequences for both genes are presented in Figure
2
. The deduced amino acid sequences matched those of the histone H2A proteins
from
T.pyriformis
(
26
) except for two conservative amino acid replacements (I
48
-> V
48
and I
138
-> L
138
; note that locations are codon positions shown in Figure
2
, with the initiator methionine codon as number 1, and not amino acid positions
in the processed protein from which the initiator methionine is removed) in
H2A1, one amino acid replacement (A
131
-> P
131
) and one inversion (T
128
S
129
-> S
128
T
129
) in H2A2. In both
T.pyriformis
and
T.thermophila
, the two H2As are highly similar except for the extreme C-termini, where the sequences are highly diverged and the H2A1 proteins are
five amino acids longer than the H2A2 proteins. The differences in H2A1 and
H2A2 within each species and the virtual identity of H2A1 and H2A2 between the
two
Tetrahymena
species argue that the gene encoding
Tetrahymena
H2A duplicated before the two species diverged and that the two genes do not
perform identical functions.
Using RNase protection, the 5'-ends of
HTA1
and
HTA2
messages were mapped to -61, -58 and -52, -51 respectively; 3'-ends of the two messages were mapped to
890 and 538-539 respectively (Fig.
3
, and summarized in Fig.
2
). We also mapped these ends using the method of RLM-RACE (
23
). The 5'-ends of
HTA1
were mapped to -60, -56 and -44; 5'-ends of
HTA2
were mapped to -49, -48 and -43. The 3'-ends of
HTA1
were at 878-886, and for
HTA2
they were at 532-534 (
23
). It seems to be a general feature for most
Tetrahymena
messages to have multiple start and stop sites (
23
). The small discrepancy between the results obtained by RNase protection and
those by RLM-RACE probably reflects the inaccuracies in measuring the real length of
protected RNA fragments on sequencing gels when DNA sequencing ladders are used
as markers (
24
). The ends mapped by sequencing the RLM-RACE products are likely to be more accurate.
HTA3
, the gene encoding the H2A.F/Z variant hv1 of
Tetrahymena
, contains two introns (
17
), and the H2A genes of some fungi (
39
) and some plants (
40
) have been reported to contain introns. The sequences of the genomic clones of
HTA1
genes indicate that there are no interruptions within the protein coding
regions. The RLM-RACE experiments show that there are no introns in the 5'- and 3'-UTRs, because the sequences of the mapped cDNA
clones are identical with those of the genomic clones. For the
HTA2
gene, the cDNA clones (pXL40 plus 5'- and 3'-RLM-RACE clones) match perfectly with the genomic
clones where both sequences are available. Within the region between the two
primers used in the inverse PCR reaction, the genomic sequences were not
cloned. However, the size of the inverse PCR product (1.9 kb) is consistent
with that of the genomic fragment on the Southern blot (2.0 kb) taking into
account the 78 bp between the two primers. This argues that no intron exists in
the
HTA2
gene either.
Northern blots of growing (dividing) and starved (non-dividing)
Tetrahymena
RNAs were hybridized with a probe recognizing both
HTA1
and
HTA2
, or with a probe recognizing
HTA3
(Fig.
4
). Both the
HTA1
and
HTA2
genes are highly expressed in growing (dividing) cells and low levels of
expression are also detectable in starved (non-dividing) cells. PhosphorImager quantitation (Molecular Dynamics,
Sunnyvale, CA) of the Northern blots indicates that the
HTA1
gene is expressed at a 55-fold higher level in growing than in starved cells, and the
HTA2
messages are 34 times more abundant in growing versus starved cells. It is not
clear whether these large differences in expression between growing and starved
cells represent extremely low levels of expression in non-growing cells or indicate that a small fraction of the cells is still
growing, perhaps slowly. The
HTA3
messages are clearly present in both growing and starved cells, although a 9-fold higher level of expression is detected in growing cells (see also
41
). Thus, the
HTA1
and
HTA2
genes are more likely to be cell cycle regulated while the
HTA3
gene encoding hv1 is a partially replication independent variant, expressed
throughout the cell cycle (
41
) and in non-growing cells.
The DNA sequences in Figure
1
establish the amino acid sequences of the H2A proteins in
T.thermophila
. The
HTA1
and
HTA2
genes encode different proteins. H2A1 is five amino acids longer than H2A2 and
the two proteins diverge considerably at the C-terminal ends. There also are three internal amino acid differences
between the two proteins. Both genes are expressed, as revealed by Northern
blot analysis, RNase mapping experiments and by the presence of
electrophoretically distinct proteins on SDS-PAGE (
42
). Whether the structural differences between the two proteins reflect any
functional differences can now be tested using the cloned genes and newly
developed methods for creating gene knockouts in
Tetrahymena
(
8
-
11
).
When viewed in their entirety, the
Tetrahymena
histone genes illustrate many of the common properties of the histone gene
superfamily of eukaryotes. In a few organisms, histone genes are present in
large numbers of tandem repeats containing one copy each of the five types.
However, in most organisms, each histone class is represented by a small number
of genes dispersed at multiple sites in the genome but with some tendency to
cluster (
1
).
Tetrahymena
shows this latter arrangement, but with minimal clustering. Thus, the only
histone genes that appear to be physically linked are
HHF2
and
HHT2
encoding an H4 and an H3 which are divergently transcribed and are separated by
~400 bp in
T.thermophila
(
12
,
15
) and in other
Tetrahymena
species (
38
,
43
,
44
).
There are three reasons why genes are duplicated and retain function (as opposed
to becoming pseudogenes): (i) to provide additional gene copies (dosage
repetition); (ii) because the duplicate genes evolve to encode proteins with
slightly different functions (variant or isotype repetition); or (iii) because
they evolve different patterns of gene expression that are selectively
advantageous (regulatory repetition). The
T.thermophila
histone gene superfamily appears to exhibit all of these phenomena. Thus, the
two H4 genes,
HHF1
and
HHF2
, encode identical proteins (
12
,
15
) and are both expressed during macronuclear S-phase, probably for dosage reasons, but only
HHF2
is expressed during micronuclear S-phase (regulatory repetition;
45
).
HHT1
and
HHT2
also encode identical proteins, while
HHT3
encodes a slightly different H3 (
7
,
13
).
HHT1
,
HHT2
,
HTA1
and
HTA2
are likely replication variants since they are expressed much more highly in
growing than in starved cells (
7
,
46
and this report), while
HHT3
is a replacement/basal or partially replication-dependent variant gene expressed in both growing and starved cells (
7
,
46
). The patterns of expression of these H2A and H3 genes in the cell cycle have
not yet been analyzed.
HTA3
, the gene encoding hv1 is a replacement/basal variant or partially replication-dependent gene (
41
and this report).
HTA3
probably also represents a case of variant repetition. It encodes an H2A.F/Z-type variant and phylogenetic analyses (
2
,
16
,
17
) have indicated that these variants diverged from the major H2A genes early in
eukaryotic evolution and have been under even greater evolutionary constraint
than the major H2A genes. It is also worth noting that the
HHF1
gene encoding one of the
Tetrahymena
H4s may have properties of both replication-dependent and of replacement/basal variants in as much as its expression
is greater in growing than in starved cells (
47
). Because both the
HTA1
and the
HTA2
genes and the
HHF2
gene (encoding another H4 protein) are present at extremely low levels in
starved cells compared with growing cells, it is not clear whether they show a
low level of basal expression or just reflect a population of cells that is
small and/or cycling slowly.
All of the
Tetrahymena
histone genes analyzed to date are transcribed into polyadenylated messenger
RNAs, a property shared with histone genes in fungi (
48
) and higher plants (see
40
, for a recent example). In multicellular animals, only basal/replacement
histone genes produce polyadenylated mRNAs. This evolutionary distribution
argues that polyadenylation of histones messages is the primitive state and
that absence of polyadenylation in the messages of replication-dependent variants in multicellular animal cells probably reflects an
evolutionarily recent loss. Also, the stem-loop structure associated with transcription termination of non-polyadenylated histone messages of multicellular animals (see
49
for a recent review) is absent in polyadenylated histone messages.
Interestingly, the messages encoding the mammalian H2A.X variant exist in both
polyadenylated and non-polyadenylated forms (
50
,
51
).
The distribution of introns in
Tetrahymena
histone genes also is illustrative. The
HHO
gene, encoding
Tetrahymena
macronuclear H1, was the first replication variant shown to contain an intron (
14
). While intron containing genes encoding replication variants are not common,
they have since been found in plants (
40
) and in fungi (
39
). In multicellular animals, introns have only been found in basal/replacement
genes. However, introns are not a universal feature of basally expressed
histone genes. In
Tetrahymena
, the basal/replacement gene (
HTA3
) encoding hv1 contains introns (
17
), while that (
HHT3
) encoding an H3.3 (formerly hv2) does not (
7
). As in the case of polyadenylation, this phylogenetic distribution makes it
likely that introns were present in histone genes before the divergence of
replication and basal/replacement variants and before the divergence of
animals, plants, protists and fungi and were lost in the evolution of
replication-dependent variants in multicellular animals.
Table 1
In summary, with the studies described here,
T.thermophila
joins
S.cerevisiae
as the only organisms whose entire (known) histone gene complements have been
cloned and sequenced (see Table
1
).
Tetrahymena
thermophila
is unique in having the only completely known histone gene complement
containing genes for H1 and minor replacement/basal H2A and H3 variants which
are present in most eukaryotes but appear to be lacking in
S.cerevisiae
(
4
). With the recent development of methods for mass transformation (
8
) and for gene replacement (
9
-
11
) in
Tetrahymena
, it should now be possible to analyze H1, and H2A and H3 variants
in vivo
using the types of molecular genetic analyses that have been used to study the
functions of major core histones in yeast.
The authors wish to thank Josephine Bowen for critical reading of the
manuscript. This work was supported by Public Health Service Grant GM-21793 from the National Institutes of Health.
Histone protein
Gene
Nuclear location
No. of References
Mac
Mic
genes
Linker histones
H1
HHO
+
-
1
(14)
Mic LH
MLH
[alpha]
-
+
[beta]
-
+
1
a
(52)
[gamma]
-
+
[delta]
-
+
H2A
H2A.1
HTA1
+
+
1
this report
H2A.2
HTA2
+
+
1
this report
hv1
HTA3
+
-/+
b
1
(16,17,53)
H2B
H2B.1
HTB1
+
+
1
(18)
H2B.2
HTB2
+
+
1
(18)
H3
H3.1
HHT1
,
HHT2
+
+
2
(7,13)
hv2
HHT3
+
-
1
(7)
H4
HHF1
,
HHF2
+
+
2
(12,15)
REFERENCES
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