RAPID AMPLIFICATION OF THE 3’-END OF THE C 4 I CODING REGION

First strand cDNA from HTCL polyA+ selected mRNA was generated by using a 3' Rapid Amplification of cDNA Ends (RACE) kit (Gibco BRL Life Technologies). A primer (UAP) complementary to the primer used for first strand cDNA synthesis (AP) and the C4^ specific oligonucleotide I generated a smeared product in PCR amplifications from HTCL polyA+ mRNA (not shown). 1% of the products from these PCR reactions were further 'nested' by using the UAP and the C4^ gene specific oligonucleotide 3 and generated a single abundant 876bp product in PCR amplifications (fig 9).

The 876bp 'nested' PCR product derived from the UAP and oligonucleotide 3 was Sall/Xbal double digested and ligated into Sall/Xbal double digested pUC19 plasmid (designated clone 3) (fig 10 d). Double stranded sequencing of clone 3 using primers complementary to pUC19 (-40 and M l3 reverse) plus oligonucleotide 2, identified that it contained coding information with absolute homology to purified C4^ microsequence data and previously sequenced clone 1.

I. SEQUENCING

Double stranded sequencing of the three C4^ clones (1,2 and 3) allowed overall alignment, translation and comparison of the encoded protein with partially purified C4^.

The complete cDNA sequence for C4^ is shown in figure 11. 58bp of 5' leader sequence can be seen preceding the first ATG (fig 11 A). This is followed by a 594bp opening reading frame (ORE) ending with a TGA stop codon (597-600) (fig 11 B (a)). Following on from the stop codon is approximately 746bp of 3' untranslated region (based on PCR product size and transcript size (results, section R. 1) of which 105bp have been sequenced (fig 11 C). The translated amino acid sequence for C4^ is shown below the open reading frame (fig 11 B (b)) and the amino acid sequence of the peptides obtained from purified C4^ are s hown below their counterparts (fig 11 B (c)).

A diagrammatic representation of the C4^ clones (1, 2 and 3) showing the oligonucleotides that generated them and the C4^ coding regions they encode is shown in figure 12.

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F IG U R E 9. Rapid amplification o f the 3'-end o f the C4^ coding region. 10% o f all PCR products were loaded and visualised on 1% agarose gels and ethidium brom ide staining.

A 1Kb ladder (Gibco BRL) is loaded (a) to indicate m olecular weight. An arrow on the

right indicates a m olecular weight o f 876bp. First strand cDNA from H TC L RNA was generated using a 3'- Rapid A m plification o f cDNA Ends (RACE) kit (Gibco BRL). A primer, (UAP) com plem entary to the prim er used for first strand synthesis (AP) and the C4^ gene specific oligonucleotide 1 generated a sm eared product from H TC L RNA in PCR am plifications (not shown). 1% o f the products from these PCR reactions were further 'nested' using the UAP and the C4^ gene specific prim er oligonucleotide 3 and

generated a single 876bp product in PCR amplifications (b and c). Note the abundance o f

product when 400ng o f H TCL poly A+ selected m RN A (b) is used in first strand

synthesis com pared to when l|ag o f H TCL total RNA is used (c). A control PCR

am plification reaction, with com ponents provided in the RACE kit, produces a single

F IG U R E 10. Cloned C4^ specific PCR products. 2p.g o f digested m iniprep products were loaded and visualised on 1% agarose gels and ethidium brom ide staining. A 1Kb ladder (Gibco BRL) is loaded to indicate m olecular w eight (a), with w eights indicated in Kb on the left. The PCR product derived from oligonucleotides 1 and 2 was blunt-end ligated into Sm al digested pUC19 plasmid and designated clone 1. D ouble digestion o f clone 1

with PstI and EcoRI restriction enzym es produces the 438bp PCR insert (b). The PCR

product derived from the dC (i3) anchor prim er and oligonucleotide 4 was X bal digested,

ligated into X bal digested pUC19 plasmid and designated clone 2. D igestion o f clone 2

with X bal restriction enzym e produces the 469bp PCR insert (c). The 'nested* PCR

product derived from UAP and oligonucleotide 3 was S all/X bal double digested, ligated into Sall/X bal double digested pUC19 plasmid and designated clone 3. Double digestion of clone 3 with Sail and Xbal restriction enzym es produces the 876bp 'nested' PCR insert

non-coding leader sequence found present in clone 2. 58 nucleotides was the largest region o f 5'-leader sequence found in all clone 2 inserts sequenced. B: H TCL C4^ nucleotide sequence obtained from overlapping R T-PCR , 5'-anchor and 3 -RACE

amplified then cloned products (a) is listed above the deduced am ino acid sequence (b)

and aligned peptides from purified H TCL C4^ (c). A putative actin-binding domain is

marked in p ink and the position of potential serine and theronine phosphorylation sites

marked in blue . The location o f two possible EF-Hand structures are marked in yellow.

Identity to a consensus nuclear localisation signal is m arked in green. Three cysteine

residues at am ino acid positions 38, 63 and 124 are circled. C : 3'-untranslated region (UTR) present in clone 3. 105 nucleotides o f 3'-UTR has been sequenced. N ote the presence o f a second in fram e TGA stop codon at position 670 confirm ing that translation cannot extend this region in this reading frame. This indicates that the cDNA sequence presented for C4^ encodes the full length protein.

C4i cDNA AND DF-RIVED PEPTIDE SEOUENCK (A) - 5 8 c a g t g c g c c g c t c t c c a g c c c g c t t g a a c g c t c c c c g c a g c c a c c g c c a c c c a t t g g a ( B) ( a ) ( b ) ( c )

ATG GCC AAC AGG GGA COT GCA TAT GGC CTG AGC AGG GAG GTG GAG ( 4 5 ) M A N R G P A Y G L S R E V Q ( 1 5 )

G P A Y G L S R ( a)

( b ) ( c)

GAG AAG ATT GAG AAA GAA TAT GAT GGA GAT GTG GAG GAG ATG GTG ( 9 0 ) Q K I E K Q Y (d a D L E Q I L ( 3 0 )

( a ) ( b ) ( c )

ATG GAG TGG ATG AGG AGG GAG TGG GGA AAG GAT GTG GGG GGG GGG ( 1 3 5 ) I Q W l l T T Q G R K D V G R P ( 4 5 ) ( a ) ( b ) ( c ) ( a ) ( b ) ( c ) ( a ) ( b ) ( c ) ( a ) ( b ) ( c ) ( a ) ( b ) ( c ) ( a ) ( b ) ( c ) ( a ) ( b ) ( c ) ( a ) ( b ) ( c ) ( a ) ( b ) ( c ) ( a ) ( b ) ( c )

GAG GGT GGA GGG GAG AAG TTG GAG AAG TGG GTG AAG GAT GGG AGG ( 1 8 0 ) Q P G R E N F Q N W L K D G T ( 6 0 )

E N F Q N W L K

GTG GTA TGT GAG GTG ATT AAT GGA CTG TAG GGG GAG GGG GAG GGG ( 2 2 5 ) V L G E L I N A L Y P E G Q A ( 7 5 )

GGA GTA AAG AAG ATG GAG GGG TGG AGG ATG GGG TTG AAG GAG ATG ( 2 7 0 ) P V K K I Q A S T M A F K Q M ( 9 0 )

K I Q A S T M A F K

GAG GAG ATG TGT GAG TTG GTG GAA GGA GGT GAG GGG TAT GGG ATT ( 3 1 5 ) E Q I S Q F Q A A E R 1 3 G I ( 1 0 5 )

Y G I

AAG AGG ACT GAG ATG TTG GAA ACT GTG GAG GTG TGG GAA GGA AAG ( 3 6 0 ) N _{È Z} T D __I _{_ -L__} T V _ D __ L _{W z k i} G K ( 1 2 0 ) N T T D l " "f Q T V D L w E G K

AAG ATG GGG TGT GTG GAG GGG AGG GTG ATG AAT GTG GGT GGG GTG ( 4 0 5 ) N M A G V Q R T L M N L G G L ( 1 3 5 )

T L M N L G G L

GCA GTA GGG GGA GAT GAT GGG GTG TTG TGT GGG GAT GGG AAG TGG ( 4 5 0 ) A V A R D D G L F S G D P N W ( 1 5 0 )

A V A R D D G L F S G D P N W

TTG GGT AAG AAA TGG AAG GAG AAT GGT GGG ;y\G, TTC TGG GAT AAG ( 4 9 5 )

F _{C “} K / P _W D N ( 1 6 5 )

F P K N F s D N

GAG GTG GAA GAG GGG AAG AAG GTG ATG GGG TTA GAG ATG GGG AGG ( 5 4 0 )

Q L Q E G K N V I G L Q M G _m ( 1 8 0 )

Q L Q E G K N V I G L Q M G T

AAG GGG GGG GGG TGT GAG GGA GGG ATG ACT GGG TAG GGG ATG GGA ( 5 8 5 )