ý.,,: V. ý ýý . - Nottingham eTheses - University of Nottingham

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* added prior to use. PEB recipe above was used in Antarctica, but PMSF and Pepstatin A were replaced with a Complete Jj protease inhibitor tablet (Roche) when the protocol was performed in laboratories in Nottingham University. 20% Buffered Glutaraldehyde Na2HPO4 - 14.145g in 500m1 (A) NaH2PO4 - 10.585g in 500m1 (B) Then: 152.5m1 A+ 97.5m1 B+ 250m1 distilled H2O = 500m1 buffer Then: 800m1 glut + 200ml Buffer = 20% Buffered gluteraldehyde Adjust Ph to 7.0 with NaOH TE Buffer (for protein and genomic DNA extractions) 10 mM Tris-HC1 1 mM EDTA Adjust pH to 8 236
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Page 1 and 2:
COLD ADAPTATION STRATEGIES AND DIVE
Page 3 and 4:
, 71 "If you march your Winter Jour
Page 5 and 6:
1.6.3. Molecular typing techniques
Page 7 and 8:
3.2.1.2.3. DOC and chlorophyll a ..
Page 9 and 10:
5.3.2.5. The Sphingomonas group, is
Page 11 and 12:
LIST OF FIGURES Figure I. I. Diagra
Page 13 and 14:
Figure 3.17a Mean bacterial abundan
Page 15 and 16:
LIST OF TABLES Table 1.1. Types of
Page 17 and 18:
List of Abbreviations AFP AFGP AFLP
Page 19 and 20:
ACKNOWLEDGEMENTS This study was fun
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Chapter 1: INTRODUCTION 1.1 - Tempe
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1999 ; Hand & Burton, 1981) or iden
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Cold adaptation is dependant on the
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not at 25°C is a function of the a
Page 29 and 30:
The most prolific literature on col
Page 31 and 32:
process known as thermal hysteresis
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dominant, role in the ice-growth in
Page 35 and 36:
explanation for this diversity is t
Page 37 and 38:
1.5.3.5 - Membrane bound solute tra
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een well established (Russell & Nic
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& Bowman, 2001; Labrenz & Hirsch, 2
Page 43 and 44:
The reaction mix contains both deox
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gene mixtures and hence can be used
Page 47 and 48:
displaying a high number of common
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AACGTCGAAA AACCTCGAAA (Organism A)
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particular set of environmental par
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Lake Maximum Depth (m) Approximate
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Larsemann Hills __ rp3rtrnent of th
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Sampling trips took two days during
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Sigma, Sigma-Aldrich Company Ltd.,
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oxidized and the sample then conver
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sample. An appropriate volume was l
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min to ensure complete extension of
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Figure 2.3 - Lane delineation image
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Figure 2.6b - Molecular weights are
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RNA 41F (5'-GCT CAG ATT GAA CGC TGG
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I min. The spin column was removed
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compiled using Seqman and any erron
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ecorded and related to the level of
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information regarding the cold adap
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50ppt to 186ppt. Thirteen were sali
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M N N 't M M \p \p 00 kf) M kf) bA
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as would normally be expected, as b
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1444 6ý L CC vý O Q O O NO u 'c z
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5 - 250 3 245 240 G (, -3 235 a 0-
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3.2.2.2.5 - Dissolved organic carbo
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d 14 12 10 14 12 7 10 8 6 4 2 0 00/
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SRP ('g L- 05 10 15 20 2 3 Depth4 5
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3.2.2.3 - Biological characteristic
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a 0 Bacterial Abundance (x 106 ml-1
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3.3 - Discussion 3.3.1 - Summer sam
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to high surface water temperatures
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chemocline (Rankin et al., 1999). M
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a 250 200 150 100 50 70 60 50 40 ý
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The temperature (Fig 3.2a) through
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et al., 1999). No melt out was obse
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increase in ammonium is probably du
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10 7 9 6 8 E ö 7 5 Co u c m c 40 w
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main pigments were chlorophylls a a
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Inorganic nitrogen (NO2, NO3 and NH
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744 -- 7 644 6 19 ü SE 400-- 4 2s
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salinity decreased from July to Sep
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a Bacterial abundance (x 106 1.1) 0
Page 127 and 128:
8 ö a N Ö oý L _> Q O O 4 0 rn O
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undoubtedly true of Club Lake. The
Page 131 and 132:
suggested that input of bacteria an
Page 133 and 134:
Pendant Lake is an unstratified, sa
Page 135 and 136:
non-AFP active peptides from inhibi
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. ter rý :. 'ý Figure 4.2 -Fryka
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-ý- ---- ,. r. __-_-. ___. r. ýý
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4.2 - Results 4.2.1 - Sampling and
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SPLAT score. It has been shown that
Page 145 and 146:
3'E t ý _d M! .PJ r rn 15 3.5 4 3
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4.3 - Discussion Of the 866 bacteri
Page 149 and 150:
arisen in very few species due to c
Page 151 and 152:
Chapter 5: BACTERIAL MOLECULAR TYPI
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Fig 5.1 - Photographs of ARDRA gel
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Coefficient: Dice Algorithm: lPGMAA
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The closest published relative for
Page 159 and 160:
identification was made based on 15
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Isolate number Closest phylogenetic
Page 163 and 164:
e included due to its small sequenc
Page 165 and 166:
5.3.2 - Identification of the AFP a
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explains the fluctuation in related
Page 169 and 170:
contamination was lower as the PCR
Page 171 and 172:
seen to a certain extent in the ARD
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American Type Culture Collection (A
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Phylogenetic cluster alignment of t
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et al., 1994) and the Antarctic SIM
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Chapter 6: BACTERIAL COMMUNITY ANAL
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6.2.1 - Detection of AFP active iso
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using the same PCR protocol as orig
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Figure 6.1 - Denaturing gradient ge
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6.2.2 - Temporal and spatial variat
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Figure 6.4c - Gel I image showing b
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Coefficient: Dice Algorithm: UPGMA
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etween Om and 2m was obviously the
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All PCR-based rRNA molecular techni
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6.3.2.3 - Cellular lysis and DNA pu
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hybrids should be equal. However, S
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contain contaminants. If the profil
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(Section 6.3.2.4.3 ) could not be r
Page 205 and 206: most dominant species in the commun
Page 207 and 208: study was characterised as M protea
Page 209 and 210: 11 species) showed AFP activity. Th
Page 211 and 212: that AFP activity could have evolve
Page 213 and 214: 7.2 - Future Work The 19 AFP active
Page 215 and 216: REFERENCES ABYZOV, S. S. (1993). Mi
Page 217 and 218: BELL, E. M. and LAYBOURN-PARRY, J.
Page 219 and 220: Symposium on Environmental Biogeoch
Page 221 and 222: Research. 5,477-493. CLESCERI, L. S
Page 223 and 224: DRICKAMER, K. (1999). C-type lectin
Page 225 and 226: antifreeze proteins from Atlantic s
Page 227 and 228: FRANZMANN, P. D. and DOBSON, S. J.
Page 229 and 230: GRIFFITH, M., ALA, P., YANG. D. S.
Page 231 and 232: IVANOVA, E. P., KIPRIANOVA, E. A.,
Page 233 and 234: Antarctic Ecosystems. G. A. Llano (
Page 235 and 236: Howard-Williams and I. Hawes). Balk
Page 237 and 238: W. (1998). Rep-PCR-mediated genomic
Page 239 and 240: putida strains from Antarctica. Mic
Page 241 and 242: NICHOLS, D., BOWMAN, J., SANDERSON,
Page 243 and 244: PEARCE, D. A. and BUTLER, H. G. (20
Page 245 and 246: (1999). Palaeohydrological modellin
Page 247 and 248: tracers of biogeochemical processes
Page 249 and 250: R. G. Landes Company, Austin, Texas
Page 251 and 252: partial 16S rDNA sequencing. Applie
Page 253 and 254: YABUUCHI, E., WANG, L., ARAKAWA, M.
Page 255: APPENDICES Al. Media Tryptic Soya A
Page 259 and 260: . + + + I I I I I I + I + + I I I I
Page 261 and 262: Ö ON N M 1 O - N 'zt "o l'" 00 C N
Page 263 and 264: 1 T 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Page 265 and 266: 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Page 267 and 268: 1 1 1 1 I 1 + I I 1 I 1 1 I 1 1 1 I
Page 269 and 270: 1 1 1 1 1 1 1 I I 1 I 1 I 1 1 I T I
Page 271 and 272: Cl M 'tt tn '. p t- 00 O\ N M O - (
Page 273 and 274: d' O - (N M 'nt V) \O N. 00 O> N M
Page 275 and 276: 1 1 T 1 1 1 T 1 1 1 1 1 1 1 1 1 1 1
Page 277 and 278: + + i i i i i i " i " . + + + + " +
Page 279 and 280: e O --ý N M t Vn 1,0 [-- 00 O> N M
Page 281 and 282: FT ce + I I r. to \, O t- 00 O\ N M
Page 283 and 284: kn 110 r- 00 Oý N M O - N M Itt vn
Page 285 and 286: , It N M "t N M Iýt N M M M M Oý
Page 287 and 288: gj a - - - - - - - - - - - - - - -
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