Analysis of the clone FG

The carp white muscle specific MyoHC gene probe FG1706 was previously isolated and restriction mapped by Gerlach et ai, (1990). The plasmid was digested with the restriction enzymes PvuH and HindUl and the three resulting fragments subcloned into pBS^ (Stratagene) plasmid according to the methods described in Chapter 1. These subclones were then partially sequenced in order to determine the region o f the MyoHC gene covered by the FG1706 subclone. Northern hybridisation analysis o f this subclone was then performed on RNA from the carp described in 5.2.2.

5.3. Results

5,3.1 Analysis of the done FG1706

When hybridised to RNA from carp subjected to the tenperature regime described in 5.2.2, the probe FG1706 showed a pattern o f hybridisation similar to that described by Gerlach et al, (1990), i.e. the probe hybridised strongest to the MyoHC RNA from carp which were sacrificed at the higher tenperatures (Figure 5.2). However, unlike the observations o f Gerlach et a l, (1990) this probe also hybridised to RNA extracted from white muscle o f carp acclimatised to 10°C for five weeks (lane 1, Figure 5.2). No hybridisation of this probe was observed on blots containing red muscle RNA from the same experiment (Data not shown).

Sequence data generated from the subclone FG1706 (Figure 5.1) revealed that this sub clone covers a region o f a carp MyoHC isoform equivalent to exons 25 to 30 in mammalian MyoHC isoforms. In all other vertebrate MyoHC gene sequences, these exons cover approximately 1020 base pairs inferring that the clone FG1706 consists o f about 1000 base pairs of exon sequence and 700 base pairs o f intron sequence. Furthermore, the region o f the gene covered by exons 27 and 28 is known to code for the S2 hinge region o f the MyoHC molecule in other vertebrates species (Jaenicke et a/., 1990; Molina et a/., 1987; Strehler et ûf/.,1986). Therefore the preferential hybridisation pattern o f the FG1706 probe to RNA extracted from warm acclimated carp observed by Gerlach et al., (1990) and m this current study could be taken as indicative that the MyoHC isoforms expressed during warm temperature acclimation differ from the isoforms expressed at cold tenperatures in the region o f the S2 hinge.

Figure 5.1

Restriction map and partial sequence of the subclone FG1706

i) Restriction map of FG1706.

Exons are numbered according to the equivalent exon in the human P cardiac MyoHC gene. The thick black lines indicate those regions of the clone which have been sequenced.

Hindm Pvun Pvun Hindm

700 b.p

K«w3S _{m m}

700 b.p

wmm

ii) The nucleic acid sequence with the deduced amino acid translation in the one letter l.U.P.A.C code is as follows. ( --- ) indicates intron sequence whilst (= = = = = ==) indicates unsequenced regions.

GACCTGCAGCCCAAGCTTGAGGGTGATCTGAAACTGGCCCAGGAGTCCAGAAGTGA D L Q P K L E G D L K L A Q E S R S D e x o n 2 5 CCTGGAGAACGAAAAACAGCAATCAGATGAGAAGATCAAAAAGTAAGCAGACATTGA L E N E K Q Q S D E K I K K [ ---i n t r o n ATGTTTGAAGTTTACACAATTACACAACATAAGTATTTGGCACATTCTACAAAACAA --- i n t r o n --- TACTTTCAAACAGGAAGGACTTTGAGATAAGTCAACTTCTCAGCAAGATTGAGGATG ---] K D F E I S Q L L S K I E D e x o n 2 6 AACAGTCTTTGGGAGCACAGCTTCAGAAGGAAAATCAAGACCTTCAGGTAAT= = = = E Q S L G A Q L Q K E N Q D L Q V = = = = = E n d o f e x o n 2 6 a n d s t a r t o f e x o n 2 7 n o t s e q u e n c e d = = 139

CAGGTGGCTGAACTCGGAGAACAGATCGACAACCTCCAGCGGGTCAAGCAGAAGCTG Q V A E L G E Q I D N L Q R V K Q K L e x o n 2 7 GAGAAGGAAAAGCGTGAATACAAGATGGAGATTGATGACTTGACAAGCAACATGGAG E K E K R E Y K M E I D D L T S N M E e x o n 27 GCTGTGGCTAAAGCAAAGGTAACATCACCTGAGGATACATAACTTTAAAGGCAACTT A V A K A K [ --- i n t r o n --- e x o n 27 TCAAAACATGTCTATATAATCTTGGATTTCTTTAGGCTAATTTAGAGAAGATGTGCC ---i n t r o n --- = = = = = = ex o n s 2 8 , 29 a n d s t a r t o f e x o n 30 n o t s e q u e n c e d = = = = AAGGCCAACAGTGAGGTGGCTCAGTGGCGAACCAAATATGAGACTGATGCCATCCAA K A N S E V A Q W R T K Y E T D A I Q e x o n 30 CGCACTGAGGAGCTTGAGGAAGCCAAGTAAATACAACAAAGCAAAGAGTTTTCAGTA R T E E L E E A K [ --- i n t r o n --- CCCTACTTTTAATATCACAAATATTAGTAA

Increasing tem perature Day 0 1 2 3 4 5 6 7 « 9 1 0 ^ -^ Lane 1 2 3 4 5 6 7 8 9 10 11 12 13

A

B

Northern hybridisation of carp total RNA from fish subjected to a daily increase in temperature with the probe FG1706

Total RNA (30pg) prepared from the white muscle of carp described as "Group A" in 5.2.2 was separated by electrophoresis and transferred to nylon membrane. RNA was extracted from the pooled muscle samples of three fish on day 0 and two fish on each subsequent day. The 1.7 Kb insert of the sub clone FG1706 was labelled with a-dCTP by random priming (Feinberg and Volgstein.,1983) and hybridised to the RNA on the membrane under high stringency conditions (65“C hybridisation temperature and washes as described in 2.2.6).The blot was then exposed to X-ray film (Fuji RX) at -70”C for 72 hours. Lanes contain RNA extracted on individual days as indicated on the figure. The water temperatures for each day of the experiment are given in Table 5.1. Prior to commencing the increase in temperature this group of fish had been maintained at 10®C for five weeks.

5.3.2. Temperature acclimation experiment 1: Changes in MyoHC gene expression