Biotech student study group needs assistance

diane s1081448 at uottawa.ca
Thu Oct 2 09:12:52 EST 1997


Hello,

My name is Diane and I am a 4th year university student in Biology. I am
part of a "Biotechnology student study group" which is working together
to answer a set of questions given to us by our professor.  Our
professor has STRONGLY suggested that we seek answers to 11 specific
questions from various sources (eg. class notes, texts, the primary
literature, friends, colleagues, professors, internet and company
catalogues). It was suggested to me, by Mary McCarty (Associate Scientific
Editor, BioTechniques), that the methods-reagents BioSci group would be
the best newsgroups to address these questions. The 10 questions follow,  

1) While travelling through the highlands of Borneo, you become aware of
a plant natural product(an alkaloid) which induces a state of higher
consciousness during exam writing. You have synthesized a large amount
of this prodcut and investigated its mode of action. It appears that it
affects on brain cells is mediated by a receptor, probably a surface
glycoprotein. You have been given the task of isolating the human gene.
How would you do it (detailed procedure/techniques)? How would you know
you had done it (correctly)?

2)The expression of a foreign protein from a eukaryotic organism in a
bacterium such as E.coli can be complicated by the differences in the
biology of the two organisms. Discuss this problem and the potential
solutions for the cloning of an EXPORTED ENZYME. If you were faced with
these problems, what possible solutions would you propose and why?
If it was necessary to express the gene in a previously unknown
organism, how would you accomplish it? How would you know you had
accomplished it?

3)Molecular genetics provides powerful tools for the study of biological
phenomena. However, the key limiting factor(assuming no limit to MRC or
NSERC funding) in this approach is our ability to isolate the underlying
genes. Describe ways in which we can oovercome this problem. In your
answer provide a description of and rationale for the techniques you
describe. Be clear to state what types of genes can be identified by each
technique.

4)RFLP analysis holds many advantages over traditional approaches for gene
mapping. Describe thses advantages and how they can be used (must
state the traditional techniques and their disadvantages). In many cases
PCR technology can now be employed to replace RFLP analysis. Compare the
advantages and disadvantages of each technique for gene mapping. Also,
explain the use of both in DNA fingerprinting (forensic work).

5)Recently a new parasitic disease, due to infection by the organism Yudo
canadiensis (fictional organism) has been discovered in the Ottawa area.
Disease seems to result from degeneration of brain cells.

In a major breakthrough, you discover  genetic evidence that resistance
to the parasite can be found in a model animal suystem, the mouse strain
U0 which is inherited as a single locus, udo (autosomal,dominant allele is
udo1) WHEN mated to strain CU. You have been given the task of isolating
the human gene. How would you do it? How would you know you'd done it?

6)You are in the transgenic plant business, producing corn plants which
are resistant to insects eg. corn borer. Various resistance genes in other
plants can be identified be genetic studies and some of these might prove
useful in corn. How would you determine which are likely to be useful and
which are not? Once you have done this and identified the best candidate,
how would you produce a resistant corn plant?

7)You have isolated and characterized a cDNA clone. It is 1.4kb long but
by Northern analysis, an mRNA of 2.3kb is detected. Since one of the
objectives of your grant application was the characterization of signals
controlling expression, you need a strategy to isolate the full lenght
cDNA. Listed belaow are several possible strategies. For each one describe
how it could be employed to reach your objective.

a. cDNA library
b. PCR and a cDNA library
c. mRNA and 5'RACE
d. mRNA and 3'RACE
e. oligonucleotides and mRNA

8)As part of your genome project oyu have sequenced ten overlapping PAC
clones encompassing 500kb from a typical eukaryote. There are several
possible routes to continue the analysis. For each one describe how it
could be employed to reach your objective.

a. Northern analysis
b. chromosome walking
c. Zoo blots
d. exon trapping
e. transformation into mutant yeast lines

9)You have cloned a full lenght cDNA from your typical eukaryote and wish
to investigate some aspects of the protein encoded by it eg. subcellular
distribution,interactions with other proteins in the cell etc. You decide
to express it in E.coli in order to get lots of the protein. There are
several possible routes to continue the analysis. For each one describe
how it could be employed to reach your objective.

a. codon usage
b. fusion proteins
c. affinity chromatography
d. regulatory sequence
e. protein TAGs

10)The isolation of DNA markers linked to the phenotype of interest is a
major step forward in the genetic dissection of the phenotype. Listed
below are several possible strategies. For each one describe how it could
be employed to reach your objective.

a. RFLPs
b. microsattelites
c. RAPDs
d. AFLPs  
e. ESTs

11)In a continuation of question 10, you have used the RAPD approach and
identified a polymorphism linked to rust resistance
(R=resistance,r=sensitivity) in wheat. You clone the band, intending to
use the cloned DNA as a probe for Southern analysis prior to positional
cloning. So you isolate DNA from several generations of plants (F1 to F3)
and probe. However, when you have finished the molecular analysis. it
seems that linkage to two different chromosomes is observed. Offer several
explanations for this result and attempt to devise a strategy to test your
hypotheses. 


Any help provided would be GREATLY appreciated. Thank you.


Sincerely,
Diane

email: s1081448 at mailbox.uottawa.ca





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