Download Sequences of the Nucleocapsid Genes from Two Strains of Avian

J. gen. Virol. (1985), 66, 573-580.
Printed in Great Britain
573
Key words: IB V/nueleocapsid/mRNA A/nueleot ide sequence
Sequences of the Nucleocapsid Genes from Two Strains of Avian Infectious
Bronchitis Virus
By M. E. G. B O U R S N E L L , * M. M. B I N N S , I. J. F O U L D S AND
T. D. K. B R O W N
Houghton Poultry Research Station, Houghton, Huntingdon, Cambs. PE17 2DA, U.K.
(Accepted 2 November 1984)
SUMMARY
cDNAs prepared from viral genomic RNA purified from two strains of infectious
bronchitis virus (IBV) (Beaudette and M41) have been cloned into pBR322. Three of
these clones, which contain the complete sequences of m R N A A for both strains,
except for the leader sequences which are only present on the subgenomic messenger
RNAs, have been sequenced using the dideoxy method. The sequences are similar for
both strains, each containing a single long open reading frame of 1227 bases which
predicts a polypeptide of molecular weight approximately 45 000. The genome position
and size of this predicted polypeptide are consistent with it being the gene for the
nucleocapsid protein. The amino acid sequence shows considerable homology with
those of the nucleocapsids of murine hepatitis virus strains A59 and JHM. The major
difference between the sequences determined for the two IBV strains is that the 3' noncoding region of the Beaudette strain contains a 184 base segment which is not present
in the M41 strain.
INTRODUCTION
Coronaviruses are enveloped viruses with a positive-stranded RNA genome of 15 to 20
kilobases (Siddell et al., 1983). The virion contains three major protein structures: the spikes or
surface projections, the membrane protein, and, associated with the RNA, the nucleocapsid
protein (Sturman et al., 1980; Cavanagh, 1981 ; Siddell et al., 1983). In infected cells a number of
subgenomic m R N A species are produced which form a nested set, with a common 3" terminus,
but extending to different lengths in the 5" direction (Stern & Kennedy, 1980a; Leibowitz et al.,
1981). It appears that, in general, only the 5' proximal region of each RNA is translated (Stern &
Kennedy, 1980b; Rottier et al., 1981; Siddell, 1983: Stern & Sefton, 1984). In the case of avian
infectious bronchitis virus (IBV), and of the murine coronavirus murine hepatitis virus (MHV),
the smallest m R N A is the most abundant and codes for a phosphorylated (Stohlman & Lai,
1979; Siddell et al., 1981 ; Stohlman et al., 1983) unglycosylated (Sturman, 1977; M acnaughton et
al., 1977) polypeptide of 50 000 to 60 000 mol. wt. (Siddell et at., 1980; Stern & Sefton, 1984). This
is the only viral protein unaffected by Pronase treatment of intact virions, and is therefore most
likely to be the nucleocapsid polypeptide (Sturman, 1977). It is also approximately the same size
as the core proteins of several other enveloped RNA virus families such as myxoviruses,
paramyxoviruses and rhabdoviruses, all of which have nucleocapsids with helical symmetry
(Lenard & Compans, 1974). The nucleocapsid of coronaviruses also appears to have helical
symmetry (Oshiro,1973).
Avian infectious bronchitis virus causes economically important disease in the domestic fowl.
However, the Massachusetts-derived strain Beaudette (Beaudette & Hudson, 1937), commonly
used in the laboratory, has had over 250 passages in eggs and is no longer pathogenic for
chickens. In contrast, the Massachusetts strain M41 has had over 400 passages in chickens and
remains pathogenic (Geilhausen et al., 1972; Collins & Alexander, 1980).
In this paper we present the sequences of the nucleocapsid genes of the IBV strains Beaudette
and M41 which have been obtained from cDNA clones of virion RNA.
0000-6431 © 1985 SGM
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574
M. E. G. BOURSNELL AND OTHERS
METHODS
cDNA chining. Three cDNA clones, derived from RNA isolated from gradient-purified virus, have been used
for sequencing. For Beaudene the oligo(dT)-primed clone C5.322 was used, which includes the poly(A) tail
derived from the 3' terminus of the viral genome and extends for 1930base pairs in the 5' direction. The production
of this clone was exactly as described in Brown & Boursnell (1984). For M41 two clones, C41.81 and 196, have
been used. C41-81 was produced as described in Brown& Boursnell(1984). The clone 196 was made by the method
of Gubler & ttoffman (1983). It was shown to include the poly(A)sequences from the 3' terminus of the viral RN A
by probing Southern blots of Pstl-digested plasmid DNA with 7-32Pkinase-labelled poly(U) (Brown & Boursnell,
1984). The positions of these clones and of the mRNAs are shown in Fig. 1.
Recloning into Ml3Jbr DNA sequencing. For C5-322 the viral insert, which had been cloned into the PstI site of
pBR32Z was isolated from acrylamide gels (Maxam & Gilbert, 1980) and recloned into SmaI-cut, phosphatasetreated, Ml3mpl0 replicative form DNA (Amersham) after digestion with DNase I (Anderson, 1981) or the
restriction enzymes AluI and Rsal, and into BamHI-digested M13mp9 after digestion with Sau3Al. For the M41
clones the plasmids were digested with DNase I and recloned into MI 3mpl0 without isolation of the viral inserts.
Clones containing viral sequences were then identified by transferring bacterial colonies grown from colourless
plaques on to nitrocellulosefilters and probing with radiolabelled viral probes (Grunstein & Hogness, 1975). Two
probes were used : ;,_3_,pkinase-labelled alkali-degraded viral RNA, and cc-3zP-labelledsingle-stranded cDNA,
reverse-transcribed from viral RNA using calf thymus primers. Using these two probes single-stranded phages
containing inserts from both the plus and minus strands of viral sequences were selected.
DNA sequencing. Sequencing was carried out by the dideoxy method (Sanger et al., 1977, Bankier & BarreU,
1983). Additional sequence information was obtained by reverse sequencing of some of the M13 clones (Hong,
1981). [ct-3sS]dATP (Amersham) was used in the sequencing reactions and buffer gradient gels were used to
analyse the reaction products (Biggin et al., 1983).
Computer analysis o[sequence data. Sequence data were read directly into a BBC microcomputer using a sonic
digitizer (Graf/Bar, Science Accessories Corporation) and the computer program READGEL, which is a version
of GELIN (Stadem 1984a). Data were analysed on a VAX 11/750 minicomputer using the programs of Staden
(1982b, t984b). The program DIAGON (Staden, 1982a)was used for the matrix comparison with the MHV JHM
nucleocapsid sequence. For comparison of the amino acid sequences a span length of 21 was used. The
'proportional' score of 234, above which a point is plotted, was selected so that only matches significant at the 1~0
level would be displayed.
RESULTS
The region of sequence presented in this paper is 1836 bases, from the C T T A A C A A A
homology sequence which occurs at the junction of the leader and body of m R N A A (Brown et
a/., 1984) to the poly(A) sequence at the 3' end. Thus, the whole of the body of m R N A A is
presented for each strain. For the Beaudette clone C5-322 this region has been completely
sequenced on both strands. For M41 this region, covered by two clones, has also been completely
sequenced on both strands, although since they overlap to some extent this did not entail
complete double-stranded sequencing of both clones. For M41 the region covered is only 1652
bases since neither M41 clone contains a sequence of 184 bases that is present in the Beaudette
clones. In Fig. 2 the top line of sequence is IBV Beaudette, with the bases that are different in
M41 marked underneath. A translation of the main open reading frame is also shown with
amino acids altered in M41 indicated.
The sequences from both strains contain a single long open reading frame of 1227 bases,
which predicts a polypeptide of molecular weight 45032 for Beaudette and 45214 for M41.
These predicted molecular weights agree fairly well with the estimated molecular weight of
5 1000 (51 K) of a polypeptide produced by translation in vitro of m R N A A (Stern & Sefton, 1984)
and the molecular weights (50K to 54K) (Macnaughton et al., 1977; Nagy & Lomniczi, 1979:
Collins & Alexander, 1980; Cavanagh, 1981 ; Stern et al., 1982) reported for the nucleocapsid
polypeptide from IBV virions. It should be noted that the predicted molecular weight does not
take into account the phosphorylation of the polypeptide, which in the case of the M H V J H M
nucleocapsid increases the apparent molecular weight of the polypeptide from 57K to 60K
(Stohlman et al., 1983). The sequences around the putative initiation codon, G U C A U G G , are
common among sequences flanking functional eukaryotic initiation codons (Kozak, 1983). The
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Sequence o / IB V nucleocapsM gene
I
M
575
N
Genome
I
t
~
~
J
~
~
5v
4
2
i
t
mRNA D
mRNA C
mRNA/3
mRNA A
.-~ C5.322
C41.81
196
3'
6kb
Fig. l, Diagram of the Y-proximal 4 kilobases of the IBV genome showing the positions of clones
C5. 322, C41-81 and 196. The gaps in clones C41-81 and 196 indicate the region of sequence present in
the Beaudette clones but absent in the M41 clones. Also shown are the positions of tuRN As A to D. The
rectangles at the top represent the main open reading frames in mRNAs A,B and C, M being the
membrane polypeptide, N being the nucleocapsid protein and 7.5K and 9.5K being the two open
reading frames at the 5' end of mRNA B (Boursnell & Brown, 1984).
coding sequences for the two strains are very similar. There are 87 single base changes, the
majority of which are purine/purine or pyrimidine/pyrimidine changes. These base changes
result in 23 altered amino acids, many of which are alterations of a conservative nature.
Examination of the amino acid sequence shows that it predicts a polypeptide which is
enriched in basic residues with an overall positive charge at neutral p H of 19 for Beaudette and
20 for M41. These basic residues are clustered in distinct regions; for example, in the Beaudette
sequence the regions from bases 727 to 791 and from 1167 to 1205. The C-terminus of the
polypeptide is, however, enriched in acidic residues, with 14 residues out o f 47 being aspartic or
glutamic acids. The Beaudette and M41 amino acid sequences also contain 28 and 32 serine
residues respectively, for which the clustering is even more marked. In the Beaudette sequence
for example there is a stretch of 28 amino acids from bases 594 to 677 which contains nine out of
the total number of 28 serine residues.
Outside the predicted coding region the major difference between the two strains is the 184
base stretch o f sequence present in Beaudette and absent in M41. That this is not a cloning
artefact is confirmed by two observations. In the case of M41 the deletion is present in the two
independently derived clones. In the case of Beaudette eight independently derived clones have
restriction maps which show that they have this stretch of sequence, all containing a HindlII site
at position 1436, which is within the 184 base region (data not shown). The deletion in M41
occurs only four bases after the 3' end of the large 1227 base open reading frame.
DISCUSSION
The main open reading frame present in these sequences predicts a polypeptide whose size
and genome position is consistent with it being the gene for the IBV nucleocapsid polypeptide.
The basic nature of this predicted polypeptide would also be expected for an RNA-associated
protein. It is very interesting to compare the sequences presented here with sequences published
for the nucleocapsid genes of another coronavirus, M H V (Armstrong et al., 1983; Skinner &
Siddell, 1983). A comparison o f the R N A sequences o f I B V Beaudette and M H V J H M (Skinner
& Siddell, 1983) has been m a d e using the matrix comparison computer program D I A G O N
(Stadenl 1982a). The R N A sequences a p p e a r to be unrelated for most of the sequence with a few
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P
D
~
G
D
N
370
A
A
E
C
T
280
W
Y
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T
8
260
G
S
G
270
V
K
P
V
W
450
V
A
Q H G Y W R R Q A R F K P G K G G R K P V
~GCAA ~TACTGGAGACGCCAAGCCAG~I'PTAAGC~AGGCAAAGGT~GAAAACCAG
G
T
300
310
320
330
340
350
360
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380
390
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t
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T I ~ I ~ G A ~ ~
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1960
3070
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~ AGAG"I~CCCAAAC'FI~.AACT A G A T f ~ C
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960
970
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990
L R/K F E F T T V V P C/R D D P Q F D N Y V K
Acq~GA~'ITGAA~ q T A C T A C T ~
CCCATGTGAIf C ~ C ~ G ~ ' f A A q T A T ~ ' f
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GT~~ACTAAAGGT~GGAGGGGAA~TGACAAGATGAATGAGGAAGGTATTAAGGATGGGC
A
820
830
840
550
860
870
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GTGT£ACAGC.AA'f~CCFA~CL'C~ACCACi~CATC~
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R S D/G S G/E D D L I A R A A K/R I I Q
A G A A G q G A T I L ~ G A TGACCITA ~~ C I ~ C C A A A G A T A A ~ " C A G G A T C
G
G
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T
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670
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700
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Q K K G S R I T K A K A D E M A H R R Y C K R T I P P
~ T G A A A T C ~ T C C ~ A q ~ I ~ C A A G C G C A C T A T C C C A C C ~ f A A T T A T A
G
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730
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750
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770
780
790
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R A P S R E G/V S
GTAGAGCACCAq~ACGTGAA
T
640
650
G P D G N F R W D F I P L N R G R S G R S T A A S S A
GCCC~CL~ ~T ~ T I ' ~ C I ' ~ C A ~ 5 ~ C ~ A G G A G ~ G A T C A A C A G C A ~ ~ A
A
T
T
T
G
550
560
570
580
590
600
610
620
A/G K G A D T K S/F R S N Q G T R D P/S D K F D Q Y P L R F S D G
C~AACK~TAC'fAAA
T~fA~TCCAATCAGGGTACAAGA~TCCTGATAAG~CCAATACCCACTA(SGATq~
G
T
T
TCT
C'9
C
T G
T A C
460
470
480
490
500
510
520
530
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~
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G S S G N A/V S W F Q A I K A K K L N T/S P
TUGG~TGCATC~GCAATAAAAGCCAAGAAG~AAATACACCTC~GTI~GGTAGCC~
T
T
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T
190
200
210
220
230
240
P
180
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M A S G K A A/T G K T D A P A P V I K
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G
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100
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~ C ~ A G T A q ' I ~ C C ~ C ~ G G A A C A C A T A C A T A A T A A T
T
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...............
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1700
ITI~AAOI~AC~AGAC PAC<~I~ T I ~ ' ~
1670
1320
1830
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T
1800
1710
1620
Fig. 2. D N A sequence of 1836 bases of IBV Beaudette c D N A clone C5,322.
Beneath the sequence are marked those bases which are different in the M41
clones. Above the sequence is a translation of the main open reading frame in
single-letter amino acid code. Where the amino acids differ between "the strains
the two residues are shown separated by a slash, the Beaudette amino acid being
the first. The region of sequence present in Beaudette but not in M41 is
underlined with a dashed line.
1810
ATAGq~fAAAAT~ATACC~AGTATAGA~ITAGACC
OCA C~CGGAGTACGA T C G A G C < Z f A C A G C A C f A G G A C C ~ L L ' C A ' F f A ~ G A G C T A A A ~ I q ' I I A [ ~ ' g ~ G ~ q ~
C
1720
1730
1740
1750
1760
1770
1780
1790
1630
CC~AC~,~q'AGACCCq q'AGA~'I~AA%~q'AG~PITAA'Iq~AG~PITA~
TAAGAG'YTAAGGAAGATAGGCATGTACC PI~]AT~AOCTA CAq~I~ fAqL~GGGAAAq~'IL~TAA%~TGTC~ A ~ A ~
A
T
1540
1550
1560
1570
1580
1590
1600
1610
,FCIq-I,~ r C ~ I
"fC~ATTGTA%~f~±%C~'I~'FAST~I~'TGATIUTCA]
R~A~P]'~ ~ A ~ A ~ T A G
................................................................................
1450
1460
1470
1480
1490
1500
1510
1520
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Gq~,ACATI-I~I~AAA
CAC£Aq'FI~ ~ I Y / ~ I q ' I ~ C ~ f A & ~ _ . A A ' I ' F A T T A C A G G C A ' ~ ' A ~ '
~ T A C ~
'
..........................................................................................
1360
1370
1380
1390
1400
1410
1420
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E
K L/P K K Q D D ~ A/V D K A L T
~TAAAC ~Aq~[~/A~GA~(IGAC~AC~C~TCCAC
C
'fG
1200
1210
1220
1230
L E F Y D E P K V I N ~ G D A/S A L G E N
AGCI~TITfATGATGAC~~GGAGAGAATGAAC~FIGAGTAACATAA'It~CX~f
G
T
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1280
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1300
1310
1320
G
Q R P K K ~
AACAC~.~G2~AC~C~
G T R P K D D E P K/R P K S R $ S S R P A T R G N S P A
TAGGAA~GATGACGAACCAAAACCAAAGqC_ACGCTCAAGTfCAAGA~ACAAGAGGAAAT~CfCCACCGCCAAGAC
A
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1100
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1130
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Sequence of IB V nucleocapsid gene
MHV amino acids
200
100
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577
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400
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Fi ;. 3. Plot from DIAGON (see Methods)of the homologiesbetween MHV JHM nucleocapsidamino
aci, sequence(across the top) and IBV Beaudette nucleocapsid amino acid sequence (down the lefthand side). Points represent matches at the 1% significance level.
short stretches of homology. However, a comparison of the predicted amino acid sequences of
the polypeptides reveals distinct similarities. Fig. 3 shows these results. A point is plotted at any
position where the similarity between two stretches of sequence is above the 1% level of
significance. The program not only looks for identical amino acids but also for residues with
similar properties. Diagonal lines of points therefore represent regions of high homology. The
highest degree of homology, represented by the most prominent diagonal line on the diagram,
occurs between amino acids 50 and 150 in the IBV sequence (85 and 185 in the MHV sequence)
and there are several other smaller regions. Fig. 4 shows the striking degree of amino acid
homology which occurs in part of this region. Another interesting similarity between the two
nucleocapsid sequences lies in the distribution of serine residues. The major cluster of serine
residues from bases 594 to 677 is mirrored almost exactly in the MHV JHM sequence (although
this is not otherwise a region of very high homology between the two sequences), and several
smaller clusters of serines also have their counterparts in MHV. This is interesting, since it is
known that the nucleocapsid protein of MHV is phosphorylated specifically at serine residues
(Stohlman & Lai, 1979; Siddell et al., 1981).
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578
M. E. G. BOURSNELL AND OTHERS
60
70
80
90
100
11 0
GSGWP~NENIKPSQQHGYWRR QAR FKI~K~VPDAWYFYYffb~3PAADLN3qG73TQDGIVWV
+--+++
-+
--+++--+++
+
-- +
++
+--
+
----
+++++
++++
+
-
++
-+--+++
GQGArPIANGIPASQQKGYWYRI ~ R R S F K T P ~ K Q L L P R I ~ P Y A G A E Y G D D I ~ q V
90"
100"
110"
12
°0
1 30"
1 40"
150"
Fig. 4. Part of the amino acid sequences of MHV JHM (top line) and IBV Beaudette (bottom line).
Identical amino acids are marked with a plus (+) and amino acids with similar properties are marked
with a minus ( - ) . The numbers show the number of amino acids from the N-terminus of the
polypeptide.
The amino acid differences between IBV Beaudette and IBV M41 are not distributed evenly
along the sequence but are restricted to certain regions. Similarly, the amino acid differences
between MHV JHM and MHV A59 are clustered and the main region of change in MHV (from
amino acids 136 to 162) is paralleled very closely by a major group of altered amino acids in IBV
from amino acids 103 to 134. It is probable that these represent part of the nucleocapsid
polypeptide which is less important to the function of the molecule.
Although there is very little homology between the RNA sequences of IBV and MHV in the
coding sequences and the 5' non-coding sequences, at one position in the 3' non-coding
sequences there is one very interesting homology (Stern, 1983). This is a 10 base match,
G G G A A G A G C T , which occurs at almost exactly the same position in the two viruses, i.e. 81
bases from the 3' end for 1BV and 82 bases from the 3' end for MHV. Elsewhere in the 3' noncoding sequences there is essentially no homology between IBV and MHV. Such a striking
homology at a particular position strongly suggests some function, possibly in replication of the
putative negative-strand replicative intermediate. This sequence does not occur in the 1BV
Beaudette leader sequence (Brown et al., 1984) but some homology between the leader and the
reverse complement of the 3' end of the IBV genome (i.e. the 5' end of the putative negative
strand) can be seen. In particular, nine bases from the 5' end of the negative strand and 24 bases
from the end of the leader there is a region in which 10 bases out of 11 match.
It is likely that most of the 3' non-coding sequence serves an important function, as it is very
highly conserved between Beaudette and M41 (three changes out of 315 bases or 99-0~o). It also
contains many repeated sequences, with the sequence A G T T T A occurring six times, and the 10
base sequence T T T A G T T T A A occurring three times. Bearing in mind the apparent
importance of the 3' non-coding region, it is interesting to speculate on the origin of the 184 base
sequence present in Beaudette and absent in M41, The base composition of this stretch of
sequence is distinct from the rest of the IBV sequence. It has a very high T content of 50 ~ which
is constant along its length, whereas that part of the 3' non-coding region present in both strains
has a variable base composition which overall is very similar to that for IBV clones sequenced to
date. When these differences are considered, and in view of the high homology between the
strains in the rest of the 3' non-coding region, it seems most likely that this sequence is an extra
piece of RNA inserted into the genome at some time in the history of the Beaudette strain.
We are grateful to Penny Gatter, Bridgette Britton, Philip Green and Anne Foulds for excellent technical
assistance. We wish to thank Dr Martin Bishop, Department of Zoology, Cambridge University, for donating a
copy of his computer program READGEL. This research was carried out under Research Contract No. GBI-201 I-UK of the Biomolecular Engineering Programme of the Commission of the European Communities.
REFERENCES
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vol. B5 : Techniques in Nucleic" Acid Bioehemisto', pp. B508, 1-34. Edited by R. A. Flavell. Ireland: Elsevier.
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Sequence o f I B V nucleocapsM gene
579
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reading frame encoded by m R N A B. Gene 29, 87-92.
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