TABLE 4.

Rescue of mutations s34 and s44 by plasmid-borne genes

Mutant	Chromosome regiona	No. of plasmids	Presumed relevant gene(s)
s34	IIL (C30B4 and C3D6)	1	wsc1 (homolog of S. cerevisiae WSC genes, which encode sensors of surface stress)b
	IR (C1006)	1	rgf2 (encodes a Rho-GEF)c
	IIICEN (C645)	2	rgf1 and rgf3 (encode Rho-GEFs)d
	IR (C1F7)	4	rho1e
	IIR (C1289)	3	pob1 (encodes homolog of S. cerevisiae Boi1 and Boi2, involved in Rho protein signal transduction)f
	IIR (C12D12)	2	pck2 (encodes a protein kinase C)g
s44	IIL (C30B4 and C3D6)	1	wsc1 (see above)h
	IIICEN (C645)	3	rgf1 and rgf3 (see above)i
	IR (C1F7)	19	rho1j

Plasmids were recovered during attempts to clone the genes containing mutations s34 and s44 (see materials and methods).

^aThe chromosome arm and relevant cosmid(s) from the S. pombe genome project are indicated.

^bThe plasmid contains the entire 1125-bp (no introns) ORF SPBC30B4.01c [designated wsc1 on the basis of the similarity of its product to the S. cerevisiae Wsc proteins (Levin 2005)] plus ∼2.1 kb of downstream flanking sequence; the 1326 bp between wsc1 and the uncharacterized ORF SPBC30B4.02c; and the C-terminal 822 bp (∼39%) of SPBC30B4.02c, which seems unlikely to be relevant. The 2.1 kb downstream of wsc1 contains no known or predicted genes.

^cThe plasmid contains the entire 3576-bp coding region (including two introns totaling 99 bp) of rgf2 (SPAC1006.06); the 729 bp between rgf2 and the divergently transcribed och1 (SPAC1006.5c); the entire 1191-bp (no introns) ORF of och1 (encoding an α-1,6-mannosyltransferase) plus 314 bp of downstream flanking sequence; the 883 bp between rgf2 and ORF SPAC1006.07; and the N-terminal 337 bp (∼29%) of this ORF (which encodes a putative translation factor). och1 and SPAC1006.07 seem unlikely to be relevant.

^dOne plasmid contains the entire 3828-bp (no introns) rgf3 (SPCC645.06c) plus ∼850 bp of upstream sequence; the 1355 bp between rgf3 and myo2 (SPCC645.05c); the N-terminal 2307 bp of the 4581-bp (no introns) myo2 ORF; but no part of rgf1 (SPCC645.07). The other plasmid contains the N-terminal 1229 bp (∼32%) of rgf3 (which does not include the Rho-GEF domain); the 1051 bp between rgf3 and the divergently transcribed rgf1; and the N-terminal 3959 bp of the 4051-bp coding region (including one 46-bp intron) of rgf1.

^eEach plasmid has one or the other of two distinct inserts, each of which contains the entire 940-bp coding region (including one 331-bp intron) of rho1 (SPAC1F7.04) plus flanking sequences. The region of overlap between the inserts contains no other known or predicted genes.

^fThe plasmids have three distinct inserts, each of which contains the entire 2811-bp coding region (including one 195-bp intron) of pob1 (SPBC1289.04c) plus flanking sequences. The region of overlap between the inserts contains no other known or predicted genes.

^gThe region shared by the two plasmid inserts contains the entire 3051-bp (no introns) ORF of pck2 (SPBC12D12.04c); the entire 933-bp coding region (including five introns totaling 291 bp) of rev7 (SPBC12D12.09, encoding a putative DNA polymerase ζ); and the C-terminal 43 bp (∼3%) and 306 bp (∼24%) of cct1 (SPBC12D12.03) and SPBC12D12.05c, respectively, which seem unlikely to be relevant. [Note that the SPBC12D12.09 ORF number is out of sequence because this gene was missed during initial annotation (V. Wood, personal communication).]

^hThe plasmid contains the C-terminal 797 bp (∼71%) of the wsc1 ORF (see footnote b) plus 1584 bp of downstream sequence that contains no other known or predicted gene.

ⁱThe three plasmids have identical inserts that contain only an internal 1930-bp fragment (lacking the N-terminal 1681 bp and the C-terminal 440 bp) of the rgf1 coding sequence (see footnote d). This fragment contains the Rho-GEF domain. In addition, the two rgf1/rgf3 plasmids isolated in attempting to clone the s34 gene (see footnote d) were also able to rescue the phenotype of the s44 strain JW405.

^jThe plasmids have at least six distinct inserts, all of which contain the entire rho1 gene (see footnote e) plus flanking sequences. The region of overlap between the inserts contains no other known or predicted genes.