The progress of some of the teams in the Network has been affected by difficulties in recruiting post-doctoral workers of eligible nationality. This means that the two year tenure of the post-doctoral workers starts later in some groups than others.
Research report
The aim of this team is to investigate the function of troponin-H. Drosophila TnH has an N-terminal region which has the sequence of tropomyosin (Tm II) and a C-terminal region which is proline rich.
1) Progress is being made towards expressing the two domains separately in order to determine the function of each in regulating the activity of Drosophila flight muscle. There have been several problems with the initial clones of TnH obtained from previous workers which has delayed expression of the fragments. These have now been overcome. The identity of the clones has been confirmed by restriction mapping and sequencing the insert ends. PCR primers have been purchased which will allow amplification of different regions of the protein: the Tm half, the proline rich region and the complete TnH. The primers were chosen so that the protein would be split at the first methionine residue within the hydrophobic extension (the third residue of the extension). This will allow the hydrophobic extension alone to be studied. By introducing unique restriction sites in to the PCR primers, each construct can readily be transferred into the pET expression system as a single cassette. The Tm-half construct is ready for transfer into the expression system.
Preparation of the full length TnH construct, which involves splicing part of the full length mTmII cDNA onto the partial TnH cDNA, has been hampered by difficulties in re-orienting the mTmII cDNA within the M13 vector. This problem may be related to the slow growth of the phenotype associated with the presence of the TmII insert and suggests that the mTmII sequence interferes with some stage of the M13 life cycle. In order to avoid this difficulty, a PCR based assembly procedure for construction of the full length TnH mRNA will also be attempted.
Dr Clayton visited Dr Sparrow, GB, twice for a week each time, in order to screen for TmII null mutants and other mutants in the TmII gene in Drosophila.
2) Lethocerus TnH differs from Drosophila TnH. Biochemical experiments showed that Lethocerus TnH behaves like TnI, the inhibitory component of many troponins. Antibody to Drosophila TnI, obtained from Dr Ferrus, ES2, reacted with Lethocerus TnH, confirming that the molecule has sequence in common with TnI. A Lethocerus cDNA expression library will be prepared and clones selected with anti-TnH in order to sequence Lethocerus TnH.
Research report
The problems encountered with the employment (only 2 months) and replacement of Dr Yacoub has limited progress. But Dr Sparrow's team has begun work on the project.
1) Characterisation of the D53 mutation recovered by the Ferrus team, ES2. D53 is a suppressor mutation of the hdp~2 mutation in the troponin-I gene. D53 maps to the 3rd chromosome region 88F, which contains two tropomyosin genes, TmI and TmII and the indirect flight muscle-specific actin gene, Act88F. PCR has been used to sequence the Act88F gene from flies homozygous for D53. There are no changes in the DNA sequence that would lead to amino acid changes in the actin.
Dr Yacoub made PCR product from genomic DNA of a wild-type strain and from D53 homozygotes using primers to conserved sequences in exons in the TmI and TmII genes. An unusual feature was the appearance of a larger than expected PCR product in the wild-type strain that was not found in the D53 strain. These PCR products will be sequenced. Examination of the original hdp~2 strain will show whether the absence of the large PCR product in D53 is due to the mutation or is a polymorphism in some strains.
2) Attempts to clone the mp35 protein. The MAC144 antibody provided by Dr Bullard, DE, which reacts with troponin-H and the mp35 protein of Drosophila has been used to isolate 16 potential cDNA clones from a Drosophila 'head' cDNA library.
3) Recovery of null mutations in thin filament protein genes. These would be suitable hosts for P-element germline transfection of flies with 'engineered' mutations. A chromosome inversion with a breakpoint within the TmI gene has been isolated from dominant flightless mutants produced by gamma-irradiation. The mutation is recessive lethal in combination with the easter~5022 deficiency (removes the TmI and TmII genes). Both the dominant flightlessness and the recessive flightlessness map to the 88F region breakpoint and are 'rescued' by copies of the TmI+ gene inserted elsewhere in the genome following P-element transfection. The effects of the mutation in TmI on muscle structure, development of the larval muscles and on the structure of the TmI gene are being investigated.
Research report
The work has been delayed by the difficulty in finding a candidate of the correct nationality for the HCM postdoctoral fellowship. Dr P. Benoit will start work in October, 1994. During the last year the ES1 team has been establishing methods to be used by Benoit.
1) Regulation of troponin T isoform expression by analysis of promotor and enhancer regions of the gene. The 5' regulatory region of the gene will be sequenced using TnT cDNA probes from the 5' end (prepared in the ES1 lab.). The promotor and conserved motifs in the sequence will be identified. The function of the regulatory region will be tested by expression in vivo using P elements and reporter genes in transformed flies. Mutants in the TnT gene from the Sparrow team, GB, will be rescued to establish the function of different domains of the protein.
2) Properties of TnT isoforms. Isoforms of TnT have been separated. A TnT cDNA for one isoform has been cloned and the protein expressed in E. coli has properties similar to the native protein. The activity of partial sequences will be studied.
TnT is phosphorylated by labelling with 32P in vivo and Ca2+ binding has been demonstrated. The Ca2+ binding sequence is being identified by removing different domains of TnT. Troponin H is also phosphorylated in vivo.
3) Stoichiometry of thin filament proteins. Marco, ES1 and Sparrow, GB have labelled proteins with 35S-methionone during myogenesis in Drosophila. The proteins in the adult muscle are separated on 1 or 2D gels and the proportions estimated from the amount of label, using the known amino acid sequences. More data are needed in this study.
Research report
1) Four kilobases of genomic DNA, immediately downstream from the troponin I (TnI) gene, have been sequenced. This region harbours two transcription units thought to be functionally related to TnI. These units encode 50 kD and 36 kD hypothetical products. The 50 kD product exhibits a primary structure compatible with a transmembrane localization. The 36 kD product shows a large number of acidic residues. RNA in situ hybridization tests indicate that the 50 kD transcript is expressed in the nervous system while the 36 kD product is abundant in the nurse cells of the gonads and the egg. Antibodies are being raised against these products.
2) The structural properties of TnI are being analysed. One mutation that suppresses the held-up mutants of TnI has been studied. In particular, attention has been focussed on a held-up mutant, hdp2, causing an Ala to Val change in exon 5 of TnI and the suppressor D3 causing a Leu to Phe substitution in exon 7. Flies expressing TnI with both alterations restore muscle structure almost to perfection. The flight performance of this genotype has been analyzed by A. Prado during a two month stay with Dr Sparrow's team in York, GB. Their average wing-beat frequency is 160 Hz while the normal control value is 220 Hz. Other suppressors resulting from mutations in the myosin heavy chain gene are being analyzed.
Research report
The aim of this team is to study the structure of flight muscle thin filaments by cryo-electron microscopy.
1) Isolation of thin filaments. The flightless Drosophila mutant Ifm(2)2 was chosen as a source of thin filaments. The mutation is in the myosin gene and specifically affects the flight muscle myosin. The flight muscles have no thick filaments which makes the isolation of thin filaments easier. A method for preparing relatively clean thin filaments from the mutant has been found. Dr Ruiz visited Drs Clayton and Bullard, DE, to obtain mutants and to learn how to dissect Drosophila and prepare thin filaments.
2) Microscopy of filaments. Conditions suitable for microscopy of the filaments were investigated. The effects of different buffers on the filaments were followed by high dose electron microscopy using negative stain and the filament composition was checked by gel electrophoresis. The effect of nucleotides (ADP, ADP-Pi and ADP-BeF3) on the structure was similar to that found for reconstituted actin filaments. Three different states of the filaments were observed with the nucleotides; ADP-Pi gave the best quality images and ADP the poorest.
The best quality actin filaments in vitreous ice are obtained with ADP-BeF3. Drosophila thin filaments will be examined in ice using this nucleotide and 3D reconstructions made of the images. The structural states of the filaments in the other nucleotides will be compared.