The presence of the RG A SQ A G heptapeptide in proteins in the transgelin
superfamily within the repeat motif has already been discussed in terms of the serine phosphorylation sites contained within it (Discussion Sections X6.1A). However, the additional presence of this heptapeptide sequence near the N-terminus of human gelsolin (but not other members of the gelsolin family) in the SI domain known to contain G-actin contact sites and constituting part of the F-actin severing domain (Way et al 1989) may possibly point to a potential role in contact with actin, but obviously must await further investigation.
Homologues of a second peptide FLKAAEDY have been found in a range of actin binding proteins. It is present in a number of tropomyosin isoforms from a range of tissue- types in different species and less conserved homologues can be found repeated through the molecules. Tropomyosin is known to be composed of a number of repetitive actin binding sites that function without exact amino acid identity (Ruiz-Opazo & Nadal-Ginard 1987). Caldesmon (83-87kDa in smooth muscle) contains a core region composed of eight thirteen amino acid repeats containing alternating basic and acidic amino acids (including the residues KKAAED). Whether these repeats are involved in the cell cycle specific association of caldesmon with microfilaments is currently unknown. Two regions very
much like this peptide are found in actobindin. Actobindin is an 88 amino acid long Acanthamoeba protein with two characterised actin binding sites (Bubb et al 1991; Vancompemolle et al 1991) containing two near identical 33 amino acid repeats (Vandekerckhove et al 1990) and is capable of inhibiting an early stage of actin polymerisation by sequestering otherwise nucléation competent dimers (Bubb et 1991). The peptide LKHAET is present near the start of each of the two repeats and is involved in
cross-linking of actobindin to the glutamic acid residue at position 100 in actin (Vancompemolle et al 1991). Mutations of residues in this peptide (and in a related peptide, LKKTET, in thymosin p4) radically alter the nature of the interaction with actin
(Vancompemolle et al 1992).
These results together constitute a cogent argument supporting the proposition that
the FLKAAEDY sequence contains residues likely to be involved in close contact with actin.
Whether it actually forms part of an actin binding site obviously depends on the sequence context of the peptide in the protein and its resultant position within the three-dimensional structure of the protein. Data from secondary structure predictions and hydropathy plots support the notion that this peptide is present in a hydrophilic stretch predicted to adopt an alpha-helical configuration and likely to be on the surface of the protein (Discussion Section 4.8 & 4.9). Mutation of residues within this peptide, in a manner analagous to those performed with thymosin p4 and actobindin (Vancompemolle et al 1992) will be necessary to definitively examine the ability of this region in transgelin to interact with actin.
A very short amino acid sequence DEAG/DESG has been widely reported in both actin and actin binding proteins (reviewed in Tellam et al 1989) and has homology to the end of the LKAAEDYG peptide, namely DEAG / DESG compared to EDYG (3/4 conserved, where D-E is a conservative change). The DEAG tetrapeptide has been shown by crystallographic analysis to be immediately adjacent to an actin binding helix in gelsolin (McLaughlin et al 1993).
Any or all of these peptide regions (FLKAAEDY; RGASQAG; positive amino acid cluster (154-161, KKAQEHKR) or negative amino acid cluster (23-28, DEELEE)) may
represent complete or partial actin binding sequences. If this is indeed the case, their distribution at distinct locations would allow transgelin to bind more than one actin residue
in separate filaments and cause them to be tightly bundled in vivo. Sucrose gradient experiments with purified transgelin under conditions identical to those in which it is able to gel actin suggest that the protein remains as a monomer and as such would require at least two actin binding sites within the single polypeptide (Shapland et al 1993).
4.13.1 Genome Analysis
Hybridisation experiments using cDNA probes directed against restriction digested genomic DNA can yield a number of different results:
(i) A single band may be detected indicating that the gene in question is probably represented as a single copy (eg. Dodemont et al 1982; Lewis & Cowan 1985; Fechheimer et al 1991).
(ii) Two bands may be detected indicating either that there are two related genes in the genome or that there is a restriction site within the single gene separating two regions recognised by the probe (eg. Scheel et al 1989; Abe et al 1990). The occurrence of an internal restriction site causing double bands is precluded by the use of at least three different enzymes in separate lanes.
(iii) Multiple bands may be detected indicating the existence of:
(a) a multiple gene family (Buckingham 1985; Dodemont et al 1982).
(b) a single gene encoded by multiple exons separated by very large introns each with potential restriction sites (Sambrook et al 1989).
(c) existence in the genome of pseudogene copies of the gene (non-functional copies with different genomic locations and restriction patterns (Macleod et al
1986; Varghese & Kronenberg 1991).
(d) a non-random distribution of restriction sites (resulting in a cluster of sites within the region recognised by the probe).
To avoid some of these problems rat genomic DNA aliquots were digested with a variety of restriction enzymes (Sambrook et al 1989) and the products fractionated by agarose gel electrophoresis and transferred to hybridisation membranes. Hybridisation of these samples with an a^^P-labelled HI cDNA insert followed by washing to low stringency (2xSSC, 0.1% SDS at 65°C) produced continuous intense labelling in all lanes covering the entire range of restriction fragment sizes. This had previously been described in a number of cases in the literature, either for probes representing repetitive satelhte DNA
sequences (Sambrook et al 1989), probes with a high purine (A or G) nucleotide content (Lewis & Cowan 1985), or short probes (Holland & Hogan 1986). Increasing stringency (IxSSC) resulted in the detection in short exposures of very weakly labelled multiple bands in all lanes (not shown). Similar staining was described as ‘low copy number abundance bands’ with a cDNA probe for a gelsolin like actin-binding protein Mbhl (Prendergast & Ziff 1991) and may in the case of transgelin suggest the existence of related genes in the genome. At this stringency some of this binding may be considered to be due to crossing to the C4^, calponin and NP25 genes, however, since hybridisation of this probe at this same stringency in Northern blots with mRNA derived from tissues likely to contain message for these proteins fails to produce any detectable signal, this possibility seems unlikely. Alternatively it is possible that the weak signal represents non specific binding to satellite bands within the genomic DNA digests (Sambrook et al 1989). Final washes to 0.2xSSC, 0.1% SDS at 65°C resulted in the detection of a single band in all of the lanes. The existence of a single band at different molecular weights depending on the restriction enzyme used very strongly suggests that transgelin is encoded by a single gene in the rat genome.
4.13.2 Evolutionary Conservation
Genomic DNAs from the indicated species were digested with EcoRI, blotted onto hybridisation membranes and hybridised with a^^P-labelled HI cDNA insert in a process commonly known as ‘Zoo-blotting’. A single strongly hybridising band is seen in human genomic DNA, this functions to confirm the findings with the rat genome of the existence of a single gene.
M ollusc genomic DNA (Aplysia spp.) was obtained from fresh tissue. Hybridisation of H I with EcoRI digested DNA yielded a single relatively strong band above a smaller very much less intense band (not shown). The appearance of two bands is most likely due to the existence of an EcoRI site within the gene in this species.
Drosophila melanogaster (oregon R) genomic DNA digested with EcoRI and hybridised to H I produced two bands above a high level of background smearing. The similar intensity of these two bands suggests that they may represent a single gene with an EcoRI site located centrally relative to the alignment of the probe or possibly hybridisation to the mp20 gene. Increasing stringency (from 2xSSC at 50°C to 2xSSC at 60°C)
functions to abolish all binding to Drosophila DNA. This may suggest either reduced sequence homology between HI and Drosophila transgelin or the inclusion of introns that reduce the extent of target sequence hybridising to each molecule of probe. The strong continuous labeling pattern seen above the bands of interest in many lanes may reflect the incomplete digestion of the genomic DNA or the presence of large unrestricted introns within the gene.
HI hybridised very strongly with a single band in Schizosaccharomyces pombe genomic DNA. Multiple minor bands were visible on over-exposed autoradiographs at higher molecular weights. These may be due to incomplete digestion of the yeast DNA or more likely to related genes present in the genome. The intensity of the hybridisation may be either a function of a high degree of sequence conservation between human and yeast transgelin or its small genome size such that there are proportionately more copies of the transgelin gene per microgram of digested DNA when compared to the human genome.
The existence of a single strongly hybridising band as far back in evolutionary history as the fission yeast S pombe correlates well with the results of inununoblot analysis of these cells. These experiments with an anti-C4 polyclonal (but not the monoclonal) antibody show the presence of a single related protein that comigrates with purified sheep aorta transgelin (Shapland et al 1993). The conservation of the transgelin gene over the 1 to 2.5 x 10^ million years since the divergence of eukaryotic single- and multi-cellular organisms provides potential evidence of the important in vivo function performed by transgelin.The existence of well characterised yeast genetics systems will present obvious opportunities for gene ‘inactivation’ experiments (eg. Magdolen et al 1988). Analysis of the function of transgelin in yeast cells may provide valuable insights into its role in normal mammalian cells and by inference the effects of its loss in transformed cells.
4.14.1 Northern Blot Analysis
Northern blot hybridisation of radiolabelled cDNA probes to immobilised preparations of total RNA allows the sensitive determination of the size and abundance of specific mRNA molecules (Sambrook et al 1989). Extracted total RNA or polyA"*" selected mRNA was fractionated in the presence of formaldehyde and immobilised on hybridisation membranes before being probed with labelled HI or Rl.