ANALYSIS OF EXONS 1A AND

The lemur DNA sample was not amplified by the exon 4, 8 and 11 primers. This was thought to reflect the level of nucleic acid divergence which has occurred between the intron sequences of these primates. Therefore, in order to investigate the level of nucleotide and amino acid conservation of PGM1 in

Figure 7.8 Multiple sequence alignments o f primate, ra b b it and ro de n t amino acid sequences from a) exon 4 and b) exon 8. Consensus sequence is fro m the human PGM7*1+ allele. The bar (-) indicates an identical amino acid.

a)

186 226

consensus EIVDSVEAYA TMLRSIFDFS ALKELLSGPN RLKIRIDAMH G humanl --- --- --- --- -- human 2 C --- chimp --- --- --- --- -- gorilla --- --- --- --- -- orangB --- --- --- --- -- orangS --- --- --- --- --

Human polymorphism: A r g 2 2 0 to Cys220

b)

382 425

consensus SDHIREKDGL WAVLAWLSIL ATRKQSVEDI LKDHWQKYGR NFFT human+ --- --- --- --- --- human- --- H - - --- chimp --- --- --- --- --- gorilla --- --- --- --- --- orangB --- --- --- --- --- orangS --- --- --- --- --- rat --- R --- -F - - -- mouse --- F - -

Figure 7.9 Autoradiograph of exon 11 nucleotide sequences from gorilla, chimpanzee and orangutan (1 8 6 2 -1 898bp). Nucleotide substitutions in the 3'UTR sequence are shown in bold.

gorilla and chimpanzee sequence

1895 A

gorilla chimpanzee orangutan (Daniel) (Masikini) (Henry)

A C G1895 T T T orangutan sequence G A G T A G 1881

Figure 7 .1 0 Multiple sequence alignm ents o f prim ate, ra bb it and ra t exon 11 nucleotide sequences.

Coding sequence is in upper case, 3' UTR is in low er case. Consensus sequence is fro m the human PGM7 *3 'UTR 1 allele. The bar (-) indicates an identical nucleotide and the asterisk (* ) indicates deletions.

1712 1761

consensus GGAGAGGACG GGACGCACTG CACCCACTGT CATCACCtaa gaagacaggc humanl --- --- --- --- --- human2 --- --- --- --- --- huitianS --- --- --- --- --- human4 --- --- --- --- --- chimp --- g - gorilla --- --- --- --- --- orangB --- --- --- --- --- orangS --- --- --- --- --- 1762 1811

consensus ctgatgtggt acgtccctcc acccccggac ccatccaagt catctgattg humanl --- --- --- --- --- human 2 --- -t--- --- --- --- human] --- -t--- a --- --- human4 --- a --- --- chimp --- --- --- --- --- gorilla --- --- --- --- --- orangB --- a --- --- orangS --- --- --- --- --- rabbit -a at-- ---****--- --- rat t g --- c -- t a a -t g cac t g c --- 1812 1860

consensus aagagcat*g acagaaacaa aatgtattca ccaagcattt taggatttga humanl --- --- --- --- --- human2 --- c --- --- human] --- c --- --- human4 --- c --- --- chimp --- --- --- --- --- gorilla --- 1 --- orangB --- 1 --- orangS --- 1 ---

rabbit ----g — —g --- —g — g — agg — c c —a c e t — g a — tg rat ---ca — ---***-c —g --- g a —e t —g c c — a c —catc

1861 1898 consensus humanl human2 human] human4 chimp gorilla orangB orangS rabbit rat

ctttttcact aaccagttga cgagcagtgc atttacaa

a - c ---acac t -a---1 -

lemur, primers designed to the coding sequences of exons 1A and 5 were used to amplify both the lemur and the great ape samples.

The samples were amplified by exon 1A primers E l F and E1R. Nucleotides 136 to 276 were sequenced from lemur, gorilla, chimpanzee, orangutan (both a Sumatran and Bornean) and man. Primers E5F and E5R were used to amplify exon 5. Sequence was obtained from nt 802 to 925 of the six primates.

The PGM1 3/7 alleles, found at polymorphic frequencies in some Asian-Pacific populations, are characterized by a mutation in exon 1 A. An A to T transition at nt 265 leads to a substitution of Lys^? encoded by A AG, for Met®^, encoded by ATG, to give rise to the 3/7 alleles. In all the primate samples tested, an A base was found at nt 265, conserving the codon A AG. Thus, Lys6?, which is

associated with the 2/1 alleles of human PGM1, is found in primates. This codon is also conserved in rabbits. Rats, in contrast, show two base changes, such that the codon ACC encodes the amino acid Thr®'^.

The exon 1A sequences of man, chimpanzee and orangutan were identical. An A to G transition was seen in gorilla at nt 269, although it was a synonymous mutation. The lemur showed greater variation in the coding sequence, with four synonymous mutations at nt 176, nt 200, nt 230 and nt 252 (Figures 7.11 and 7.12). A missense mutation was also identified, at nt 214, an A to T

transversion giving rise to an amino acid substitution of Gln^o to Leu^o. This missense mutation is also seen in rat.

In exon 5, man, chimpanzee and gorilla nucleotide sequences were identical. A synonymous mutation at nt 815 was identified in orangutan. The lemur

exhibited four base changes (Figure 7.13), of which two at nt 866 and nt 872 were synonymous. The first missense mutation was at nt 900, a G to C transversion leading to the substitution of Glu^79 to Gln^79^ and the second at nt 911, a T to G transversion leading to the substitution of Phe282 to Leu282. Unexpectedly, in rabbit, rat and mouse PGM1, despite a number of nucleotide substitutions in exon 5, the amino acid sequences are identical to man (Figure 7.14).

Figure 7.11 Autoradiograph of exon 1A nucleotide sequences from lemur, gorilla, chimpanzee, orangutan and human (1 7 1 -2 0 8 b p ). Nucleotide substitutions between the lemur and other primates are shown in bold.

lemur G A T C G A T C G A T C G A T C G A T C other primates

sequence ^ \ ^ sequences G \ ^ / G 220 T — r 220 A / _ \ A oo 176 A A J J

lemur gorilla chimpanzee orangutan human (Columbo) (Daniel) (Katja) (Blossom)

Figure 7.12 Multiple sequence alignm ents o f prim ate, ra bb it and ra t exon 1A sequences a t the a)DNA level and b) amino acid level. Consensus sequence is from the human PGM1*1+ allele.

The bar (-) indicates an identical nucleotide o r amino acid.

a)

1 3 6 1 8 5

consensus GGGTGAÀGGT GTTCCAGAGC AGCGCCAACT ACGCGGAGAA CTTCATCCAG huitianA --- --- --- --- --- humanB --- --- --- --- --- chimp --- --- --- --- --- gorilla --- --- --- --- --- orangS --- --- --- --- --- orangB --- --- --- --- --- lemur --- T --- rabbit --- A --- -T-- --- 1 8 6 2 3 5

consensus AGTATCATCT CCACCGTGGA GCCGGCGCAG CGGCAGGAGG CCACGCTGGT humanA --- --- --- --- --- humanB --- --- --- --- --- chimp --- --- --- --- gorilla --- --- --- --- --- orangS --- --- --- --- --- orangB --- --- --- --- --- rabbit --- C --- 2 3 6 2 7 6

consensus GGTGGGCGGG GACGGCCGGT TCTACATGAA GGAGGCCATC C humanA --- --- --- --- -- humanB --- T --- -- chimp --- --- --- --- -- gorilla --- A --- -- orangS --- --- --- --- -- orangB --- --- --- --- -- lemur --- A --- --- -- rabbit --- A --- --- --- -- rat T --- --- T — C — C C --- — b) 2 5 7 0

consensus VKVFQSSANY AENFIQSIIS TVEPAQRQEA T L W G G D G R F YMKEAI humanA --- --- --- --- --- humanB --- --M--- chimp --- --- --- --- --- gorilla --- --- --- --- --- orangB --- --- --- --- --- orangS --- --- --- --- lemur --- L ---- --- --- rabbit --- T - - --- --- --- ---

Human polymorphism: LysG? to Met67

Figure 7 .1 3 Autoradiograph of exon 5 nucleotide sequences from lemur, gorilla, chimpanzee, orangutan and human ( 8 5 5 - 9 1 2bp). Nucleotide substitutions between the lemur and other primates are shown in bold.

lemur sequences oo human lemur (Columbo)

gorilla chimpanzee orangutan (Daniel) (Jane) (Kibriah)

other primates sequences

Figure 7 .1 4 Multiple sequence alignm ents o f prim ate, rabbit and ra t exon 5 sequences a t th e a)DNA level and b) amino acid level. Consensus sequence is fro m the human PG M ^*^+ allele.

The bar (-) indicates an identical nucleotide o r amino acid.

a)

8 0 2 8 5 1

consensus CGGCAGTTAA CTGCGTTCCT CTGGAGGACT TTGGAGGCCA CCACCCTGAC

human --- --- --- --- --- chimp --- --- --- --- --- gorilla --- --- --- --- --- orangB --- T ---- --- --- --- orangS --- T ---- --- --- --- lemur --- --- --- --- --- mouse —A — T — G — T — C — C --- T ~ --- ---- T — C --- 8 5 2 9 0 1 consensus CCCAACCTCA CCTATGCAGC TGACCTGGTG GAGACCATGA AGTCAGGAGA human --- --- --- --- --- chimp --- --- --- --- --- gorilla --- --- --- --- --- orangB --- --- --- --- --- orangS --- --- --- --- --- lemur --- C --- C --- C- 9 0 2 9 2 5 consensus GCATGATTTT GGGGCTGCCT TTGA human --- --- --- chimp --- --- --- gorilla --- --- --- orangB --- --- --- orangS --- --- --- 10mu r --- G --- --- rabbit --- C — C --- mouse --- C --- --- b) 2 4 7 2 8 6 consensus AVNCVPLEDF GGHHPDPNLT YAADLVETMK SGEHDFGAAF human --- --- --- --- chimp --- --- --- --- gorilla --- --- --- --- orangB --- --- --- --- orangS --- --- --- --- rabbit --- --- --- --- rat --- --- --- --- mouse --- --- --- ---

7.4 SUMMARY

i) The gorilla, chimpanzee and orangutan samples all carry the C at nt 723 and T at nt 1320 characteristic of the PGM 1*1+ protein phenotype seen in man. Therefore, the primate isozyme which appears to be PGM 1*1+ on lEF also exhibits the P G M ^^+ characteristics at the DNA level.

ii) The gorilla and chimpanzee samples all carry the nucleotides associated with the +++ haplotype (allele 1) of the 3' UTR polymorphism observed in man. In orangutans, at nt 1788, the two polymorphic nucleotides, characteristic of the +-+ and +++ haplotypes seen in man, were observed, but they were not

demonstrated to be polymorphic; the G nucleotide was confined to the Sumatran and the A nucleotide to the Bornean orangutans.

iii) In the three exons 4, 8 and 11 where multiple, presumedly unrelated samples of each species were investigated, no polymorphic nucleotide substitutions leading to changes in the amino acid sequence were demonstrated in the coding sequence of PGM1. The only nucleotide

polymorphism identified was in exon 4 at nt 707 in the orangutans. The three Bornean orangutans were all found to be heterozygous, whilst one of the Sumatran orangutans, Henry, was homozygous for the A nucleotide, and the other, Annie, was homozygous for the G.

iv) In exons 1A and 5, a greater level of nucleotide and amino acid divergence was evident in the lemur. The amino acids were completely conserved in the gorilla, chimp and orangutans, whilst the lemur contained one amino acid

substitution in exon 1A and two in exon 5. Interestingly, in exon 5, although the rabbit and rodent show a greater level of nucleotide diversity than the lemur, the amino acid sequence is identical to the great apes and man.

7.5 CONCLUSIONS

The molecular basis of the PGM1*^ + allele in man is conserved in the great apes which suggests that the PGM 7*1 and the PGM 7*+ alleles are conserved among primates of the hominoidae superfamily. This conclusion also provides support that the PGM 7*1+ is the ancestral allele in man. Conservation of the PGM 7*1 allele is retained in rabbits and rodents, although the PGM 7*+ allele is not.

Nucleotide sequence data from exon 11 of PGM1 in the great apes supports the proposal of the +++ haplotype of the 3'UTR polymorphism being ancestral. Although no polymorphisms were detected at the three sites in the primates, the two polymorphic bases at nt 1788 in man, G and A, are observed in the two populations of orangutans. Whether these two populations are indeed

polymorphic at this locus, or whether these mutations are fixed cannot be determined with the limited number of samples available. However, two suggestions emerge from this data. First, the nucleotide substitutions in man and orangutans may have occurred independently. Alternatively, if the orangutans are polymorphic at this locus, it may be inferred that of the three polymorphic sites in man, this was the initial polymorphism, and it occurred prior to the divergence of orangutans and man.

The lEF data provides evidence of intraspecific variation with an additional cathodal band present in the chimpanzee Halfpenny and the gorilla Daniel. DNA sequence analysis of exons 4, 8 and 11 of PGM1 in Halfpenny identified no heterozygous nucleotides, indicating the mutation which underlies this

polymorphism is not located in these exons. In Daniel, exons 1 A, 4, 5, 8 and 11 of PGM1 were sequenced. Again, no heterozygous nucleotides were identified. Therefore, this data suggests that the molecular basis of intraspecific variation in the primates occurs at a site distinct from those in man.

Exons 1A and 5 from PGM1 of lemur show a greater number of nucleotide changes compared to human PGM1, than the great apes, reflecting the

evolutionary distance between the species. The lemur belongs to the suborder prosimii, whereas man, gorilla, chimpanzee and orangutan belong to the

suborder anthropodiea. The divergence of these two suborders is estimated to have occurred between 65 and 56 million years ago. Since a number of

nucleotide changes were demonstrated in the coding sequence of lemur PGM1, the intron sequences would be expected to show even greater nucleotide divergence from human PGM1. Therefore, the failure of the PGM1 intron sited primers to amplify exons 4, 8 and 11 is most probably due to mismatches between the primers and the template DNA.

CHAPTER EIGHT:

EVOLUTION OF THE PHOSPHOHEXOMUTASES

Phosphoglucomutases (PGM) and phosphomannomutases (PMM) have been cloned from a wide variety of organisms, including prokaryotes and eukaryotes, protozoans and metazoans. Comparison of orthologous sequences

(divergence following spéciation) from these species would allow a

phylogenetic tree to be constructed, from which the evolution of the species can be inferred. However, in this chapter, knowledge of the evolution of the species is used to investigate the molecular evolution of the phosphohexomutases. Therefore both orthologous and paralogous sequences (divergence following duplication) have been included in the analysis.

Immunological studies using anti-rabbit PGM polyclonal antibodies (Chapter Three) and the low stringency and degenerate primer PGR approaches

(Chapter Four) suggest that the genes encoding the PGM2 and PGM3

isozymes are not as closely related to PGM1 as was first thought from simple comparison of isozyme patterns. Therefore, the primary aim of this

investigation was to place PGM, PMM and related sequences within an

evolutionary framework and to see if there are divergent clusters of sequences that may suggest alternative pathways of evolution for PGM2 and PGM3. Identification of these pathways may provide additional information, such as conserved protein motifs, which may lead to the identification and

characterization of these loci.

In addition, the phylogenetic analysis should identify duplications of the common ancestral gene and allow the evolutionary relationship between the many prokaryotic sequences to be investigated. Finally, the possibility of

Agrobacterium tumefaciens PGM having arisen by divergence following a

trans-kingdom horizontal gene transfer (an example of a xenologous sequence) will be examined through its phylogenetic relationship to eukaryotic and

prokaryotic sequences.