In Genetic Analysis 14.1 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 (see Genetic Analysis 14.2 for definition) of the yeast genes?

The bacteriophage ϕX174 has a single-stranded DNA genome of 5386 bases. During DNA replication, double-stranded forms of the genome are generated. In an effort to create a restriction map of ϕX174, you digest the z-stranded form of the genome with several restriction enzymes and obtain the following results. Draw a map of the ϕX174 genome.

Pstl     5386                PstI + PsiI      3078, 2308
Psil     5386                PstI + DraI      331, 1079, 3976
Dral    4307, 1079      PstI + DraI      898, 1079, 3409

To further analyze the CRABS CLAW gene (see Problems 19 and 20), 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.

EcoRI                   11.0, 3.0                     XbaI     4.5, 9.5
EcoRI + XbaI        4.5, 6.5, 3.0               XhoI     13.2, 0.8
EcoRI + XhoI        10.2, 3.0, 0.8             SalI       6.0, 8.0
EcoRI + SalI          6.0, 5.0, 3.0              HindIII   12.0, 1.5, 0.5
EcoRI + HindIII      9.0, 3.0, 1.5, 0.5

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?

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 (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.

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.