3. Lecture Flashcards
describe viral NA
- Relatively small (3-300 kilobase),
- easy to handle early genetic investigations ( phage)
- dsDNA, ssDNA, dsRNA, ssRNA
- linear or circular
- continuous or segmented
- usually haploid (diploid = retrovirus)
NA deficiency
incomplete or defective virions
cannot multiplicate in host cell
examples of alien nucleic acids
Polyomaviruses, pestiviruses: host cell nucleic acid
Arenaviruses: ribosomes
Retroviruses: onc, src genes
steps of NA purification
proteinase K enzyme digestion + phenol + chloroform + isoamylalcohol: → extraction
→ cf. → precipitation with ethanol
→ cf. → pure nucleic acid
NA purification by chromatography or protein lysis
- filters, gels, beads… to attach virus
* quicker, simpler, less dangerous, more effective
what is needed for transcription during viral multiplication
viral enzyme
+ sense ssRNA
5’ -> AAA-3’
e.g. picornavirus, act as mRNA and can thus be translated into protein of host cell
(infectious/virus prod. = +sense ssRNA)
(baltimore IV)
- sense ssRNA
3’ -> 5’
is complimentary to mRNA and thus have to be converted into + sense ssRNA by RNA polymerase before translation
= not infectious by itself
e.g. orthomyxovirus, paramyxovirus
(baltimore V)
what is observed of NA by electron microscope?
linear or circular NA
continous NA thread is found in e.g.
picornaviridae
segmented genome is found in e.g.
reoviridae
endonucleases
• bacterial defense enzymes
• recognize certain sequences
• sticky end and a blunt end
dsDNA
function of enzyme cleavage|
more exact size
– smaller fragments → handling
– agarose-gel electrophoresis
physical mapping
-> localization of cleavage sites
partial digestion with endonucleases
Molecular cloning of viral DNA:
Propagation of virus DNA fragments in bacterial plasmids
– Mass DNA production: quicker, cheaper
– Expression plasmids: protein production
– Safe bacteria are needed
