Restriction sites in Plasmodium falciparum

In search for improvement of our current YAC end rescue protocols I asked Herve Tettelin to generated a table containing all possible hexamers and their frequencies in P. falciparum genome, using our current 1,300.000 sequence reads for chromosome 2. One very useful information we can derive from this table is the occurance of a particular restriction enzyme recognition sequence and its representation in the P. falciparum genome, enabling us to select frequent and rare cutters for different purposes. An immediate application of this data is the selection of enzymes that cut well Pf DNA for YAC end rescue (Riley's method, Nucleic Acids Res. 18:2887-2890, 1990). While working at the human genome project I used to use Alu I and Rsa I and would rescue 95% of human YAC ends. For P. falciparum I have tried Alu I, Rsa I, Dra I, Asn I, Ssp I and Swa I. So far I was able to rescue 14 out of 24 ends with enzymes selected from our hexamer table data. Since I think this is a very useful information to assist in the YAC end rescue protocols and other applications, I want to share it with the group. I have listed 30 enzymes with frequencies from 1 site every 300 bp to 6-8 kb. See attachment Enz/Pfalc/Hex. I will generate a table with rare cutter sequences and their frequencies and share it too. The column "occurance" means how many sites that particular hexamer appears in the genome and "average cut" means its frequency, supposing an even distribution in the 1,300.000 bp. Hope this information will assist other groups in the isolation of terminal sequences from YACs,

Restriction.xls

MANUFACT	ENZYME	SEQUENCE	OCCURANCE	AVER. CUT	ENDS	ISOSCHIZOMER	HEAT INACT	TEMPERATURE
GIBCO	Acc I	GTATAC	323	4,319	cohesive	n.a.	resist	55
"	Afl II	CTTAAG	270	5,167	cohesive	Bfr I	part. res.	37
"	Alu I	AGCT	247	5,648	blunt	n.a.	sens.	37
BMB	Asn I	ATTAAT	2,852	489	cohesive	n.a.	?	?
GIBCO	Bcl I	TGATCA	384	3,644	cohesive	n.a.	res.	50
GIBCO	Bgl II	AGATCT	160	8,718	cohesive	n.a.	res.	37
GIBCO	Cla I	ATCGAT	152	9177	cohesive	yes/ >1	part. res.	37
GIBCO	Dra I	TTTAAA	3,539	394	blunt	Aha III	sens.	37
GIBCO	EcoR I	GAATTC	254	5492	cohesive	n.a.	part. res.	37
GIBCO	EcoR V	GATATC	202	6,906	blunt		sens.	37
GIBCO	Hinc II	GTPyPuAC	n.a.	n.a.	blunt	Hind II	sens.	37
GIBCO	Hae III	GGCC	54	25,833	blunt	yes/ >1	res.	37
GIBCO	Hind III	AAGCTT	207	6,739	cohesive	n.a.	res.	37
Pharmacia	Hpa I	TTAAC	more	more	cohesive	?	?	?
Pharmacia	same	TTAACA	887	1,573
"	same	TTTAAC	645	2,163
"	Mbo I	GATC	168	8,303	blunt	yes/ >1	sens.	37
GIBCO	Mun I	CAATTG	207	6,739	cohesive	n.a.	?	?
BMB	Msc I	TGGCCA	40	34,874	blunt	BalI,Mlu N I	sens.	37
GIBCO	Mse I	TTAA	more	more	cohesive	Tru91 I	part. res.	37
GIBCO	same	TTTTAA	4,063	343
"	same	TTAAAA	3,890	359
"	same	TTTAAA	3,539	394
"	Nde I	CATATG	776	1,798	cohesive	?	?	?
Pharmacia	Sfu I (Asu II)	TTCGAA	208	6,707	cohesive	?	?	?
BMB	Nsp V	same	208	6,707	cohesive	yes/ >1 Sfu I	part. res.	50
GIBCO	Pst I	CTGCAG	64	21,796	cohesive	n.a.	part. res.	37
GIBCO	Rsa I	GTAC	206	6,772	blunt	Csp 6	sens.	37
GIBCO	Sal I	GTCGAC	37	37,702	cohesive	n.a.	res.	37
GIBCO	Taq I	TCGA			cohesive	TthHB8 I	res.	65
GIBCO	"	TCGAA	215	6,488
		TTTCGA	214	6,518
		ATCGA	211	6,611	other possible	sites with	smaller freq.
GIBCO	Sna BI	TACGTA	200	6,975	blunt	?	?	?
GIBCO	Vsp I	ATTAAT	2,852	489	cohesive	yes/ >1	res.	37
GIBCO	Ssp I	AATATT	4,595	304	blunt	?	?	?
BMB	Swa I	ATTTAAAT	more	more	blunt	?	?	?
BMB	same	ATTTAA	2,911	479
	same	TTAAAT	2,820	495

Last Updated: 30/7/97

Analyst/Contact: Robert Huestis