Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.280030
Title: Studies of mouse actin genomic clones
Author: Begg, Carolyn E.
ISNI:       0000 0001 3453 8800
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1987
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
This thesis describes studies of two genomic clones lmA14 and lmA36, which had been isolated from a mouse genomic lambda library using a rat skeletal actin muscle cDNA probe, and which electron microscopic heteroduplex analysis had shown to contain a similar, although not identical self-hybridising (foldback) structure adjacent to the actin-like region. The objective of these studies was to determine the extent of the similarity between lmA14 and lmA36, and the nature of the actin-like DNAs and the DNA constituting the foldback structures. Detailed restriction maps of lmA14 and lmA36 were constructed in order to compare these clones. This was achieved by a combination of the following techniques : (i) single restriction enzyme digestion, (ii) hybridisation of a 32 P-labelled actin probe to the products of single and double restriction enzyme digestion, (iii) partial restriction enzyme digestion followed by hybridisation to a 32P-labelled oligonucleotide complementary to the cohesive end of the short arm of bacteriophage lambda, (iv) generation of subclones covering most of the mouse DNA inserts in lmA14 and lmA36, and subjecting these to single and double restriction enzyme digestion. The resulting maps showed that over a region of 11.0kb there were 25 restriction endonuclease sites which appeared to be identical in the two clones and 11 which were clearly different, after allowing for an extra inserted 0.5kb of DNA in lmA36 that was also found by electron microscopy. This suggested that clones lmA 14 and lmA36 contain at least 11. 0kb of similar but not identical DNA, and this suggestion was supported by the positive cross hybridisation of fragments from the two clones and partial nucleotide sequence determination of the DNA near the left and right-hand extremities of the apparent similarity. Comparison of these and other sequences from lmA14 and lmA36 indicated an average difference of 5.7%. This suggests that the two sequences diverged from a common ancestor 2.6 MY ago. Partial nucleotide sequencing was used to determine the nature of the actin-like DNA in clones lmA14 and lmA36. The portion of actin-like DNA sequenced in lmA14 corresponds to that specifying amino acids 1 to 302. Predicted amino acids at the N-terminal end of this sequence identified this as being related to the g-cytoplasmic member of the six mammalian isoforms of actin. The partial sequence of the actin-like gene of lmA36 showed it to be related to a cytoplasmic B- or g-actin, although lack of sequence at the N-terminal end prevented more precise identification. The actin-like gene of lmA14 contained a significant number of differences in predicted amino acid sequence from g-actin, and several termination codons. Furthermore it lacked introns. These features indicate that lmA14 contains an actin pseudogene of the processed type. This also appeared to be truncated at its 5' end. Comparison of the nucleotide sequence with that of a mouse g-actin cDNA clone showed 5% difference, suggesting a relatively recent origin. As lmA14 and lmA36 had similar restriction maps over much of the foldback region, the structure of this foldback was analysed in the single clone, lmA14. Areas of the three subclones thought to contain the stem of the foldback structure were sequenced, and homologous regions were identified in each subclone, that could account for the electron microscopic features. These were a region of at least 1.5 kb, adjacent to the actin, orientated in one direction (designated LH) and two regions of 1.3 kb and at least 1.0 kb, respectively (RH1 and RH2) orientated in the opposite direction. The sequence of the two regions RH1 and RH2 had an overlap of approximately 460bp. The region RH1 is outwith the DNA included in the smaller clone, lmA36, and this and the overlap of RH1 and RH2 adequately account for the electron microscopic differences of lmA14 and lmA36 in regions where they have similar restriction maps. To determine the nature of the sequences constituting the stem of the foldback element a 32P-labelled fragment of this DNA was hybridised to digested mouse chromosomal DNA subjected to agarose gel electrophoresis and transferred to nitrocellulose. The strength of the hybridisation indicated that the stem sequence was repetitive and, against a background smear, discrete bands were observed, the length of which were similar to those of the previously characterised LIMd, mouse middle repetitive DNA family. The sequences of the foldback area of lmA14 were compared to that of a recently published 'full-length' LIMd DNA sequence, confirming that the stem DNA of the foldback loop is composed of LIMd sequence. The foldback structure in lmA14 is composed of specific regions of three LIMd LINE members. One LIMd member (LIMd-LH), was contiguous with the truncated 3' end of the actin pseudogene of lmA14 and formed the left-hand arm of the stem. The right-hand arm was formed from two LIMd members (LIMd-RHl and LlMd-RH2), which are located approximately 5.2 and 11.0kb respectively to the right, of the left-hand member, in the opposite orientation. The left-hand LIMd member is at least 3.3kb in length with its 5' end contiguous with actin DNA at a position approximately 100bp from its expected 3' end. The sequence of the 3' end of the left-hand LIMd member was not determined but hybridisation with a probe containing the extreme 3' end of a different g-actin sequence, located the displaced 3' end of the actin pseudogene to a particular subclone, at least 3.1kb from the 5' end of LIMd-LH. Thus LIMd-LH has inserted independently into the g-actin pseudot gene. This measurement, together with the known length of a complete LIMd member and the presence of an internal deletion of 2. 4kb in LIMd-LH, indicated that LIMd-LH most likely contains intact 5' and 3' ends. LlMd-RH2 appears to be truncated at both ends, whereas LIMd-RHl is truncated at only the 3' end. LIMd-RHl and LIMd-LH possess several features in common which differ from the prototype full-length LIMd member. These include the same 5' end containing 1 2/3 copies of a 208bp tandem repeat, and a common 42bp insertion, and suggest the possibility of gene correction at some stage of their existence. The results presented do not allow an unequivocal decision as to whether the similar regions in lmA14 and lmA36 are the result of a gene duplication or amplification event, although indirect considerations favour the former possibility. However it is possible that the LIMd members identified in this work played a role in the original duplication or amplification of this large region of the mouse genome.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.280030  DOI: Not available
Keywords: Actin proteins and genes
Share: