The uses of multiplex detection methodologies are dramatically increasing as a means to increase sample throughput and to demonstrate quantitative differences between multiple targets in gene or protein expression analysis. In this study, we investigate the application of multiplex fluorescent detection for three proteins on the same Western blot using a laser-scanning imaging system, the Bio-Rad Molecular Imager FX. We show that independent detection and quantitation of multiple targets is achievable with little or no correction for fluorescent crosstalk by using fluorescent tags preferentially excited with different laser lines and detected at wavelengths that minimize fluorescence crosstalk. We demonstrate that the use of fluorescent detection methods can provide a tenfold greater quantifiable range but with two- to fourfold less sensitivity than chemiluminescent detection methodologies. Two examples of three-color multiplex detection using FITC-, Cy3- and Cy5-conjugated probes on Western blots are provided to demonstrate applications of this approach.

BRCA1, a breast and ovarian cancer susceptibility locus, has been isolated and maps to 17q21. A physical map of the BRCA1 region which extended from the proximal boundary at D17S776 to the distal boundary at D17S78 was constructed and consists of 51 sequence tagged sites (STSs) from P1 and YAC ends, nine new short-tandem repeat (STR) polymorphic markers, and eight identified genes. The contig, which spans the estimated 2.3 Mb region, contains 29 P1s, 11 YACs, two BACs, and one cosmid. Based on key recombinants in two linked families, BRCA1 was further localized to a region bounded by D17S1321 on the proximal side and D17S1325 on the distal side. Within this estimated 600 kb region, the contig was composed completely of P1s and BACs ordered by STS-content mapping and confirmed by DNA restriction fragment fingerprinting.

Synechocystis 6701 phycobilisomes contain phycoerythrin, phycocyanin, and allophycocyanin in a molar ratio of -2 :2 :1, and other polypeptides of 99-, 46-, 33 .5-, 31 .5-, 30 .5-, and 27-kdaltons . Wild-type phycobilisomes consist of a core of three cylindrical elements in an equilateral array surrounded by a fanlike array of six rods each made up of 3-4 stacked disks. Twelve nitrosoguanidine-induced mutants were isolated which produced phycobilisomes containing between 0 and 53% of the wild-type level of phycoerythrin and grossly altered levels of the 30 .5-and 31 .5-kdalton polypeptides . Assembly defects in these mutant particles were shown to be limited to the phycoerythrin portions of the rod substructures of the phycobilisome . Quantitative analysis of phycobilisomes from wild-type and mutant cells, grown either in white light or chromatically adapted to red light, indicated a molar ratio of the 30.5-and 31 .5-kdalton polypeptides to phycoerythrin of 1 :6, i .e., one 30 .5-or one 31 .5-kdaltons polypeptide per (a 8)6 phycoerythrin hexamer. Presence of the phycoerythrin-31 .5-kdalton complex in phycobilisomes did not require the presence of the 30 .5-kdalton polypeptide . The converse situation was not observed . These and earlier studies (R . C. Williams, J . C. Gingrich, and A. N. Glazer . 1980. J. Cel l Biol. 85 :558-566) show that the average rod in wild type Synechocystis 6701 phycobilisomes consists of four stacked disk-shaped complexes : phycocyanin (aft) 6 -27 kdalton, phycocyanin (a,8)6-33 .5 kdalton, phycoerythrin (a,8)6-31 .5 kdalton, and phycoerythrin-30 .5 kdalton, listed in order starting with the disk proximal to the core.

The tricylindrical core of Synechocystis 6701 phycobilisomes is made up of four types of allophycocyanin-containing complexes: A, (alpha AP beta AP)3; B, (alpha AP beta AP)3 .10K; C, (alpha APB1 alpha AP2 beta AP3).10K; D, (alpha AP beta AP)2.18.5K.99K; where AP is allophycocyanin, APB is allophycocyanin B, and 10K, 18.5K, and 99K are polypeptides of 10,000, 18,500, and 99,000 daltons, respectively. The 18.5K polypeptide is a hitherto unrecognized biliprotein subunit with a single phycocyanobilin prosthetic group. The tricylindrical core is made up of 12 subcomplexes in the molar ratio of A:B:C:D: of 4:4:2:2. Complexes C and D act as terminal energy acceptors. From these results and previous analysis of the bicylindrical core of Synechococcus 6301 phycobilisomes [14,15] it is proposed that the two cylinders of the Synechocystis 6701 core, proximal to the thylakoid membrane, each have the composition ABCD, and that the distal cylinder has the composition A2B2.

Abstract. Two herbicide-resistant strains of the cyanobacterium Synechococcus sp. pee 7002 are compared to the wild-type with respect to the DNA changes which result in herbicide resistance. The mutations have previously been mapped to a region of the cyanobacterial genome which encodes one of three copies of psbA, the gene which encodes the 32 kDa Qb-binding protein also known as DI (Buzby et at. 1987). The DNA sequence of the wild-type gene was first determined and used as a comparison to that of the mutant alleles. A point mutation at codon 211 in the psbAl coding locus (TTC) to TCC) results in an amino acid change from phenylalanine to serine in the DI protein. This mutation confers resistance to atrazine and diuron at seven times and at two times the minimal inhibitory concentration (MIC) for the wild-type, respectively. A mutation at codon 211 resulting in herbicide resistance has not previously been described in the literature. A second point mutation at codon 219 in the psbAl coding locus (GTA to ATA) results in an amino acid change from valine to isoleucine in the D 1 protein. This mutation confers resistance to diuron and atrazine at ten times and at two times the MIe for the wild-type, respectively. An identical codon change conferring similar herbicide resistance patterns has previously been described in Chlamydomonas reinhardtii. The atrazine-resistance phenotype in Synechococcus sp. pee 7002 was shown to be dominant by plasmid segregation analysis.