What’s in store? Mutation: Where do they come from? Mutation: How do we get rid of them? Cancer:...

November 15, 2000 All the news that's fit to transcribe

Snake swallows fifty pound ratGristle all that remains, say astounded onlookers

(RICHMOND) Type I DNA MTases differ markedly from Type II in both structure andmechanism of action (). Most notably, the former consist of two distinct subunits, a catalyticsubunit (M, encoded by hsdM) and a subunit (S, encoded by hsdS) responsible for specificbinding to DNA. The addition of a third subunit (R, encoded by hsdR) confers upon thecomplex the additional ability to cleave DNA unmethylated at the target recognition site.Type I DNA MTases have been divided into four families, IA through ID, originally on thebasis of antigenic crossreactivity.

The possibility that DNA replication, modulated by a solitary MTase, might regulatedifferentiation within filaments of Anabaena PCC 7120 motivated us to screen the strain forgenes encoding such enzymes. Unusual features in the deduced amino acid sequences ofprotein encoded by two of the genes found led us to examine them more closely within thecontext of current understanding of structure/ function relationships.

was isolated from Anabaena PCC 7120 (henceforth referred to as Anabaena) grown onBG11, modified as previously described (), by vortexing with glass beads in phenol ().Plasmids bearing expressed DNA MTase genes avaMV, dmtB/ avaMVII, anddmtD/ avaMIX were isolated essentially according to the protocol of Kiss et al. (). In brief,Anabaena DNA partially digested with Sau3AI to an apparent average size of 2 to 3 kbwas ligated with pBluescript II KS+ (Stratagene). The ligated DNA was electroporatedinto E. coli strain Gm4715 (dam- mcrB-; ref. Error! Reference source not found.), forcloning of avaMV, or K803 (hsdS3 mcrB; ref. ), for cloning of dmtB and dmtD.Approximately 400,000 colony forming units were incubated in 4 ml LB, 1 hr, then 100ml LB + 50 µg/ ml ampicillin, 6 hrs. Plasmid was isolated through a Qiagen 100 tip.Approximately 1 µg plasmid DNA was digested for at least 6 hrs with excess restrictionenzyme (DpnII for avaMV, HaeIII for dmtB, and BsrFI for dmtD). The DNA wasprecipitated, and 20% of it was used to electroporate the same strain of E. coli toampicillin resistance. For dmtB and dmtD, this was sufficient to isolate several coloniescarrying the gene. For avaMV, it was necessary to go through a second round ofamplification, starting with several hundred colonies scraped off of the transformationplate.

The gene encoding DmtA was isolated by PCR amplification using the primers 5'-ATATCATCAGGTGATCGCGC-3' and 5'-TTTGGCGCTGGGATAGTACC-3'. The geneencoding M.AvaIII and its downstream open reading frame (ORF) were isolated on thesame PCR-amplified fragment, using the primers 5'-CAGTATGCTTCAGGGGGAAA-3'and 5'-GTTGTTGATGCTTTGAGCGA-3'. Both sets of primers were derived from

Dozens remain missingfrom genetics examPuzzled officials hold out hope as search continues(RICHMOND) Routine plasmid isolation and manipulations followed standardprocedures. The presence of small DNA digestion fragments was assessed byelectrophoresis on 3% gels made with Metaphor (FMC). DNA was prepared for pulsed-field gel electrophoresis as previously described ()Analysis of DNA and proteinsequences Plasmid DNA purified through a Qiaprep minispin column was sequenced by theUniversity of Chicago DNA Sequencing Facility. Most of the DNA, and all regions wherethere were any ambiguities, were sequenced from both strands. ORFs were identified andtranslated using EditBase, provided by Niels Nielsen (Purdue University). Proteinsequences were aligned by ClustalX () and displayed by ClustalInColor, a locally writtenprogram that facilitates the coloring of amino acids to accentuate relationships betweengroups of sequences. In sequence comparisons, an amino acid was judged to be similar tothe consensus amino acid if the entry of the pair in BLOSUM62, a matrix of amino acidsimilarities (), gives a positive value. Groups of amino acids were judged to be similar toeach other if they fell into one of the following sets defined by positive values inBLOSUM62 with respect to the first listed amino acid: DEN, EDKQ, HNY, LIMV, SANT,QEKR, YFHW.

DNA MTases and restriction endonucleases were found by searching GenBank withGapped BLAST () and searching other databases listed below (last searched June 2000).DNA sequences from Synechocystis PCC 6803 and preliminary DNA sequences fromAnabaena were obtained from CyanoBase (www.kazusa.or.jp/ cyano/ cyano.html).Preliminary DNA sequences from Nostoc punctiforme ATCC 29133 (henceforth calledNostoc punctiforme), and Enterococcus faecalis V583 were obtained from the DOE JointGenome Institute (http:/ / spider.jgi-psf.org/ JGI_microbial/ html/ nostoc_homepage.html), and from The Institute for Genomic Research (http:/ / www.tigr. org), respectively. DNA MTase sequences were identified in genomic sequences bythree means: annotation ofthe sequence, annotation in REBASE (reference andhttp:/ / rebase.neb.com), a depository of information regarding restriction endonucleasesand DNA MTases, and BLAST searches using the following archetypical sequences, witheach followed by its class (Error! Bookmark not defined.,Error! Bookmark not defined.)and accession/ enzyme number: M.EcoBI (IA; rebase| 3381), M.EcoEI (IB; rebase| 3386),M.EcoR124I (IC; rebase| 3392), M.StySBLI (ID; rebase| 3571), M.Hae III (II-5mC;sp| P20589), M.Dpn IIA (II-N6mA ; sp| P04043), M.Nla III (II-N4mC ; pir| XYNHAL),M.HinFI (II-N6mAß; rebase| 3429), M.Pvu II (II-N4mCß; gb| AAA96336), M.EcoPI (III-N6mAß; sp| P08763), M.Pst I (II-N6mA ; rebase| 3483). An ORF was judged to encode aDNA MTase if it matched a positively identified DNA MTase with a BLAST score betterthan 10-4. Unidentified open reading frames were searched for known motifs usingEMOTIF (reference and http:/ / dna.stanford.edu/ identify/ ).Pl Zasmid DNA isolated from the dam- dcm- strain E. coli Gm48 (Error! Reference sourcenot found.) was hydrolyzed by a procedure modified from that described by Gehrke et al.(). About 50 µg DNA in 100 µl deionized water was denatured by boiling for 5 minutes,and plunging the microfuge tube into ice water. 200 µl 30 mM sodium acetate pH 5.2 and10 µl 20 mM zinc sulfate was added, followed by 6 units of nuclease P1 (Roche). After 6hrs of incubation at 37°, 40 µl of 0.5 M Tris (not adjusted for pH) and 5 units shrimpalkaline phosphatase (Roche) was a

Genet i cs N ews

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What’s in store?

Mutation: Where do they come from?

Mutation: How do we get rid of them?

Cancer: The result of mutation

Thanksgiving

Genetic variation

Genetics of populations

2nd UR Symposium on Agrobacterial genetics

Exam IV

What’s in store today?

Is mutatagenesis random or induced?

• Luria-Delbrück fluctuation experiment• Lederbergs’ replica plating experiment

What mutations arise spontaneously?

What is the cause of spontaneous base substitutions?

• Spectrum of mutations in lacI

• Tautomerization• Transitions vs transversions

Nature of mutationLuria-Delbrück fluctuation experiment

Culture growing without lactose

Culture able to metabolize lactose

+ lactose

Person sensitive to smallpox Acquired immunity

+ virus

Culture sensitive to bacteriophage

+ phage Acquired hereditary immunity

+ phage

Culture sensitive to bacteriophage

sensitive cellsrare mutants

Clonal growth of random mutants

vs.

Nature of mutationLuria-Delbrück fluctuation experiment

Culture sensitive to bacteriophage

+ phage Acquired hereditary immunity

+ phage

Culture sensitive to bacteriophage

sensitive cellsrare mutants

Clonal growth of random mutants

vs.

How to distinguish the two?

Nature of mutationLuria-Delbrück fluctuation experiment

Little variation from tube to tube

Great variation from tube to tube

Add phage

Add phage

SQ2: What if tubes pooled?

Add phage

Tube 1 Tube 2 Tube 3 Tube 4

Tube 1 Tube 2 Tube 3 Tube 4

4 mutants 5 mutants 5 mutants 6 mutants

Nature of mutationLederbergs’ replica plating experiment

Acquired hereditary immunity Random mutation

How to get lacI mutants

Single cell

Single colony

Culture

PGal+ Colonies

Isolate DNA

Sequence lacI

SQ6: Why a single colony?

SQ8: What kind of mutations do you expect?

What kind of mutations did Crick et al see?

Problem set 4: Mutation of Factor VIII gene Wild-type 5'-GGAGTTGAGTCATGGACTCTAAGCAGCGATCCACAAAG...

Individual a 5'-GGAGTTTAGTCATGGACTCTAAGCAGCGATCCACAAAG...Individual b 5'-GGAGTTGAGTCATTGACTCTAAGCAGCGATCCACAAAG...Individual c 5'-GGAGTTGAGTCATGGACTCTTAGCAGCGATCCACAAAG...Individual d 5'-GGAGTTGAGTCATGGACTCTAAGCAGCTATCCACAAAG...Individual e 5'-GGAGTTGAGTCATGGACTCTAAGCAGCGATCCACTAAG...

What kind of mutation has been cropping up all semester?

Base substitution

rIIA-

rIIA+

proflavin

Single base insertion/deletion

What kind of mutations were in your T4 tester strains?

Big deletions

What kind of mutations were actually observed in lacI?

GTCTGGCTGGCTGGCTGGC

GTCTGGCTGGCTGGCWild type

DuplicationSQ9See

anything unusual?

GTCTGGCTGGC

GTCTGGCTGGCTGGCWild type

Deletion

SQ6: Why start with a single colony?

Single cell

Single colony

SQ6: Why start with a single colony?Consider the alternative

Concentration? 2·109 cells/mlNumber of cells transferred? > 2·106 cells

Number of mutants transferred? > 4 mutantsNumber of mutants after regrowth of culture? > 4000 mutants

Tautomerization of bases

C TG A

C* T*

A G

Mutagenesis from tautomeric base

Mutagenesis from tautomeric base

Effects of transition mutationsTable 1: The Genetic Codea

T C A G TTT Phe(F) TCT Ser(S) TAT Tyr(Y) TGT Cys(C) TTC Phe(F) TCC Ser(S) TAC Tyr(Y) TGC Cys(C) TTA Leu(L) TCA Ser(S) TAA STOP TGA STOP

T

TTG Leu(L) TCG Ser(S) TAG STOP TGG Trp(W) CTT Leu(L) CCT Pro(P) CAT His(H) CGT Arg(R) CTC Leu(L) CCC Pro(P) CAC His(H) CGC Arg(R) CTA Leu(L) CCA Pro(P) CAA Gln(Q) CGA Arg(R)

C

CTG Leu(L) CCG Pro(P) CAG Gln(Q) CGG Arg(R) ATT Ile(I) ACT Thr(T) AAT Asn(N) AGT Ser(S) ATC Ile(I) ACC Thr(T) AAC Asn(N) AGC Ser(S) ATA Ile(I) ACA Thr(T) AAA Lys(K) AGA Arg(R)

A

ATG Met(M)b ACG Thr(T) AAG Lys(K) AGG Arg(R) GTT Val(V) GCT Ala(A) GAT Asp(D) GGT Gly(G) GTC Val(V) GCC Ala(A) GAC Asp(D) GGC Gly(G) GTA Val(V) GCA Ala(A) GAA Glu(E) GGA Gly(G)

G

GTG Val(V) GCG Ala(A) GAG Glu(E) GGG Gly(G)

Effects of transversion mutationsTable 1: The Genetic Codea

T C A G TTT Phe(F) TCT Ser(S) TAT Tyr(Y) TGT Cys(C) TTC Phe(F) TCC Ser(S) TAC Tyr(Y) TGC Cys(C) TTA Leu(L) TCA Ser(S) TAA STOP TGA STOP

T

TTG Leu(L) TCG Ser(S) TAG STOP TGG Trp(W) CTT Leu(L) CCT Pro(P) CAT His(H) CGT Arg(R) CTC Leu(L) CCC Pro(P) CAC His(H) CGC Arg(R) CTA Leu(L) CCA Pro(P) CAA Gln(Q) CGA Arg(R)

C

CTG Leu(L) CCG Pro(P) CAG Gln(Q) CGG Arg(R) ATT Ile(I) ACT Thr(T) AAT Asn(N) AGT Ser(S) ATC Ile(I) ACC Thr(T) AAC Asn(N) AGC Ser(S) ATA Ile(I) ACA Thr(T) AAA Lys(K) AGA Arg(R)

A

ATG Met(M)b ACG Thr(T) AAG Lys(K) AGG Arg(R) GTT Val(V) GCT Ala(A) GAT Asp(D) GGT Gly(G) GTC Val(V) GCC Ala(A) GAC Asp(D) GGC Gly(G) GTA Val(V) GCA Ala(A) GAA Glu(E) GGA Gly(G)

G

GTG Val(V) GCG Ala(A) GAG Glu(E) GGG Gly(G)