BackTo further analyze the CRABS CLAW gene, you create a map of the genomic clone. The 11-kb EcoRI fragment is ligated into the EcoRI site of the MCS of the vector shown in Problem 18. You digest the double-stranded form of the genome with several restriction enzymes and obtain the following results. Draw, as far as possible, a map of the genomic clone of CRABS CLAW.
What restriction digest would help resolve any ambiguity in the map?
To estimate the number of cleavage sites in a particular piece of DNA with a known size, you can apply the formula N/4ⁿ where N is the number of base pairs in the target DNA and n is the number of bases in the recognition sequence of the restriction enzyme. If the recognition sequence for BamHI is GGATCC and the phage DNA contains approximately 48,500 bp, how many cleavage sites would you expect?
We designed a screen to identify conditional mutants of S. cerevisiae in which the secretory system was defective. Suppose we were successful in identifying 12 mutants.
Based on your knowledge of the genetic tools for studying baker's yeast, how would you clone the genes that are mutated in your respective yeast strains? What is an approach to cloning the human orthologs of the yeast genes?
We designed a screen to identify conditional mutants of S. cerevisiae in which the secretory system was defective. Suppose we were successful in identifying 12 mutants.
Describe the crosses you would perform to determine the number of different genes represented by the 12 mutations.
You have isolated a genomic clone with an EcoRI fragment of 11 kb that encompasses the CRABS CLAW gene (see Problem 18). You digest the genomic clone with HindIII and note that the 11-kb EcoRI fragment is split into three fragments of 9 kb, 1.5 kb, and 0.5 kb.
Restriction enzyme sites within a cDNA clone are often also found in the genomic sequence. Can you think of a reason why occasionally this is not the case? What about the converse: Are restriction enzyme sites in a genomic clone always in a cDNA clone of the same gene?
You have isolated a genomic clone with an EcoRI fragment of 11 kb that encompasses the CRABS CLAW gene. You digest the genomic clone with HindIII and note that the 11-kb EcoRI fragment is split into three fragments of 9 kb, 1.5 kb, and 0.5 kb.
Does this tell you anything about where the CRABS CLAW gene is located within the 11-kb genomic clone?
In a typical PCR reaction, describe what is happening in stages occurring at temperature ranges
(a) 92-26 °C
(b) 45-65 °C and
(c) 65-75 °C
You have identified a 0.80-kb cDNA clone that contains the entire coding sequence of the Arabidopsis gene CRABS CLAW. In the construction of the cDNA library, linkers with EcoRI sites were added to each end of the cDNA, and the cDNA was inserted into the EcoRI site of the MCS of the vector shown in the accompanying figure. You perform digests on the CRABS CLAW cDNA clone with restriction enzymes and obtain the following results. Can you determine the orientation of the cDNA clone with respect to the restriction enzyme sites in the vector? The restriction enzyme sites listed in the dark blue region are found only in the MCS of the vector.
We usually think of enzymes as being most active at around 37°C, yet in PCR the DNA polymerase is subjected to multiple exposures of relatively high temperatures and seems to function appropriately at 65–75°C. What is special about the DNA polymerase typically used in PCR?
Traditional Sanger sequencing has largely been replaced in recent years by next-generation and third-generation sequencing approaches. Describe advantages of these sequencing methods over first-generation Sanger sequencing.
Given your knowledge of the genetic tools for studying Drosophila, outline a method by which you could clone the dunce and rutabaga genes identified by Seymour Benzer's laboratory in the genetic screen.
How is fluorescent in situ hybridization (FISH) used to produce a spectral karyotype?
You have generated three transgenic lines of maize that are resistant to the European corn borer, a significant pest in many regions of the world. The transgenic lines (T₁ in the accompanying table) were created using Agrobacterium-mediated transformation with a T-DNA having two genes, the first being a gene conferring resistance to the corn borer and the second being a gene conferring resistance to a herbicide that you used as a selectable marker to obtain your transgenic plants. You crossed each of the lines to a wild-type maize plant and also generated a T2 population by self-fertilization of the T1 plant. The following segregation results were observed (herbicide resistant : herbicide sensitive):
Explain these segregation ratios.
How would you clone a gene that you have identified by a mutant phenotype in Drosophila?
Bacterial Pseudomonas species often possess plasmids encoding genes involved in the catabolism of organic compounds. You have discovered a strain that can metabolize crude oil and wish to identify the gene(s) responsible. Outline an experimental protocol to find the gene or genes required for crude oil metabolism.