Gene Cloning

460 Gene Cloning
Protein
addition of “tags” to, 264, 274–275
aggregation of, 261
disulfide bonds in, 263, 265
N-glycosylation of, 265–266
production from cloned genes, 249–277
purification, 274–275
redox state of, 263, 265
Protein A, 301–304
Protein expression
in E. coli, 252–265, 267
in insect cells, 273
in mammalian cell cultures, 270–273
in transgenic organisms, 372–374
in yeast, 268–270
proteomics, 356–361
Protein family, 232, 235
Protein fingerprinting, 358–359
Protein fusions see fusion protein
Protein identification, 358–359
Protein sequencing, 359–361
Protein turn-over, 358
Protein-protein interactions, 297–304
detecting with co-immunoprecipitation,
301–302
detecting with TAP-tagging, 302–304
detecting with two-hybrid methods,
297– 301
Proteins, expression of, 249–277
Proteolytic fingerprint, 358–359
Proteome, 450g
Proteomics, 356–361, 450g
Pseudogene, 16
Pseudomonas aeruginosa, 149, 171, 209, 241
Pull-down assays, 301–304, 353
Pyrosequencing, 197–202
Q
QRT-PCR see quantitative real-time reverse
transcriptase-polymerase chain reaction
Quantitative real-time reverse transcriptasepolymerase
chain reaction (QRT-PCR), 76,
327–329
R
RACE see rapid amplification of cDNA ends
Rapid amplification of cDNA ends (RACE),
323–325
Rattus norvegicus, 20
Reading frame, 208–211, 450g
Real-time PCR, 76
see also quantitative real-time reverse
transcriptase polymerase chain reaction
Recombinant, 450g
Recombination, 450g
Recombination, homologous, 159, 282–284,
382–383, 398–403
Recombination, non-homologous
use in constructing gene deletions, 382–383
Recombination, site-specific, 382–383, 403–405
Red hair, 416
Regular expression, 235, 450g
Regulation of gene expression, 315–361
Repeat sequence
plants, 23
Repetitive DNA, 9, 13–15, 23, 31, 87, 90, 93, 156,
163, 411–415
Reporter gene, 335–337, 376–377, 450g
Reporter gene assays, 264, 326, 334–338,
347–349, 376–377, 379
deletion analysis, 347–350
example of use, 337
promoter probes, 334–335
reporter proteins, 335–337
site-directed mutagenesis, 347–350
vectors, 338
Reporter protein, 264, 295, 334–337, 450g
Repressor, 316, 339, 347–349, 451g
Resistance gene, 156–159
Restriction endonuclease, 451g s see also
restriction enzyme
Restriction enzyme, 38–40, 47–51, 451g
4bp recognition site, 49
6bp recognition site, 47
blunt ends, 51
compatible ends, 49
frequency of recognition sites, 47–48
function, 38–40
isoschizomers, 49
naming, 38
partial digestion, 90
rare cutters, 51
recognition sequences, 48
sticky ends, 39, 40–41
type II, 38
use in gene cloning, 38–40, 47–51
Restriction fragment, 451g
Restriction fragment length polymorphisms, 125,
161–163, 451g
Restriction map, 58–59, 451g
Retrovirus, 27
Reverse northern dot-blot analysis, 332–334,
354
Reverse transcriptase, 27, 103, 318, 327, 357
RFLP see restriction fragment length
polymorphisms
Ribosome binding site, 209, 210, 258–259
RISC see RNA-induced silencing complex
RNA interference, 287–290, 374, 375, 451g
small interfering RNA, 287