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CorrectionsBIOCHEMISTRY. For the article ''Interaction of RNA polymerase with forked DNA: Evidence for two kinetically significant intermediates on the pathway to the final complex,'' by Laura Tsujikawa, Oleg V. Tsodikov, and Pieter L. deHaseth, which appeared in number 6, March 19, 2002, of Proc. Natl. Acad. Sci. USA (99, 3493-3498; First Published March 12, 2002; 10.1073͞ pnas.062487299), the authors note the following concerning RNA polymerase (RNAP) concentrations. No correction was made for the fraction of RNAP (0.5) that is active in promoter binding. With this correction, the values of K 1 and K app (but not K f ) would increase by about a factor of 2. The relative values would remain essentially unchanged. Also, the legends to Figs. 2, 3, and 5 contain errors pertaining to the symbols used for data obtained with and without heparin challenge, the duration of the challenge, and the concentration of added heparin. The figures and the corrected legends appear below. Fig. 2. Determination of equilibrium affinities by titration of wt Fork with RNAP. The reactions contained 1 nM wt Fork and variable amounts of RNAP as shown and were analyzed by electrophoretic mobility shift immediately (OE; data shown are averages of three independent experiments) or after a challenge with 100 g͞ml heparin for 10 min (F; data shown are averages of four independent experiments). The curves shown reflect the simultaneous errorweighted fits of the data to Eqs. 3 and 4 -7. The parameters are shown in Table 1 (line 1). www.pnas.org͞cgi͞doi͞10.1073͞pnas.013667699 Fig. 3. Kinetics of complex formation. RNAP (65 nM) and wt forked DNA (1 nM) were incubated for various time intervals and then complex formation was determined immediately (Ϫheparin) or after a 2-min challenge with 100 g͞ml heparin (ϩheparin). The Ϫheparin data (s) were fit (error-weighted) with Eq. 8 with a 2 ϭ 0 (kaϪ ϭ 0.10 Ϯ 0.01 s Ϫ1 ) and the ϩheparin data (OE) with both single (k aϩ ϭ 0.036 Ϯ 0.004 s Ϫ1 ; thin line) and double-exponential (ka 1 ϭ 0.044 Ϯ 0.002 s Ϫ1 ; ka 2 ϭ (5 Ϯ 3) ϫ 10 Ϫ4 s Ϫ1 ; thick line) equations. Fig. 5.Comparison of the kinetics for formation and dissociation of competitor-resistant complexes between RNAP and wt Fork. Association data were obtained as described in the text and the legend for Fig. 3 except the concentration of forked DNA was 10 nM. Dissociation kinetics were obtained by challenging with 100 g͞ml heparin a mixture of RNAP and forked DNA that had been incubated for 30 min. The curves represent double-exponential fits of the data to Eq. 10. (A) wt RNAP. The observed association rate constants (s) are shown in the legend for Fig. 3; for the slow phase of the dissociation of the wt Fork-wt RNAP complex (F), kd 2 ϭ (1.3 Ϯ 0.2) ϫ 10 Ϫ4 s Ϫ1 . (B) YYW RNAP. The slow phase of the association reaction (F) has a ka 2 ϭ (1.1 Ϯ 0.3) ϫ 10 Ϫ3 s Ϫ1 ; the slow phase of the dissociation reaction (s), a kd 2 ϭ (6 Ϯ 1) ϫ 10 Ϫ4 s Ϫ1 . Fig. 6. BCL-6 preferentially binds to the wild-type exon 1 in Ly1 cells. Both Ly1 and the control Ly7 cells wer...

Vertebrate photoreceptor cells are the basic sensory apparatus of the retina, capable of converting the energy of absorbed photons into neuronal signals. The proximal portions of mammalian photoreceptor outer segments are synthesized daily by cell bodies, and outer segment tips are shed with a circadian rhythm, resulting in a complete turnover of outer segments about every 9 days. The shed outer segments are phagocytosed by adjacent retinal pigment epithelial (RPE) cells, and metabolites are recycled to photoreceptors. The Royal College of Surgeons (RCS) rat is a widely studied, classic model of recessively inherited retinal degeneration in which the RPE fails to phagocytose shed outer segments, and photoreceptor cells subsequently die. We have used a positional cloning approach to study the rdy (retinal dystrophy) locus of the RCS rat. Within a 0.3 cM genetic inclusion interval, we have discovered a small deletion of RCS DNA that disrupts the gene encoding the receptor tyrosine kinase Mertk. The deletion includes the splice acceptor site upstream of the second coding exon of Mertk and results in a shortened transcript that lacks this exon. The aberrant transcript joins the first and third coding exons, leading to a frameshift and a translation termination signal 20 codons after the AUG. The concordance of these and other data indicate that Mertk is probably the gene for rdy. Our results provide genetic evidence for an essential role of a receptor tyrosine kinase in a specialized form of phagocytosis and suggest a molecular model for ingestion of outer segments by RPE cells.

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by production of autoantibodies and complex genetic inheritance. In a genome-wide scan using 85,042 SNPs, we identified an association between SLE and a nonsynonymous substitution (rs10516487, R61H) in the B-cell scaffold protein with ankyrin repeats gene, BANK1. We replicated the association in four independent case-control sets (combined P = 3.7 x 10(-10); OR = 1.38). We analyzed BANK1 cDNA and found two isoforms, one full-length and the other alternatively spliced and lacking exon 2 (Delta2), encoding a protein without a putative IP3R-binding domain. The transcripts were differentially expressed depending on a branch point-site SNP, rs17266594, in strong linkage disequilibrium (LD) with rs10516487. A third associated variant was found in the ankyrin domain (rs3733197, A383T). Our findings implicate BANK1 as a susceptibility gene for SLE, with variants affecting regulatory sites and key functional domains. The disease-associated variants could contribute to sustained B cell-receptor signaling and B-cell hyperactivity characteristic of this disease.

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We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (kappa) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 V kappa genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.

To identify differentially expressed genes in multiple sclerosis, microarrays were used in a stringent experimental setting-leukapheresis from disease-discordant monozygotic twins and gene expression profiling in CD4(+) and CD8(+) T-cell subsets. Disease-related differences emerged only in the CD8(+) T-cell subset. The five differentially expressed genes identified included killer cell lectin-like receptor subfamily B, member 1, also known as natural killer receptor protein 1a/CD161, presented by the International Multiple Sclerosis Genetics Consortium as one of the non-MHC candidate loci. Flow cytometric analysis on peripheral blood of healthy donors and patients with multiple sclerosis and rheumatoid arthritis confirmed an upregulation of CD161 at the protein level, showing also a significant excess of CD161(high)CD8(+) T cells in multiple sclerosis. This subset prevalently included chemokine (C-C motif) receptor 6(+), cytokine-producing, effector-memory T cells with proinflammatory profiles. It also included all circulating interleukin-17(+)CD8(+) T cells. In the CD161(high)CD8(+) subset, interleukin-12 facilitated proliferation and interferon-γ production, with CD161 acting as a co-stimulatory receptor. CD161(+)CD8(+)CD3(+) T cells producing interferon-γ were part of intralesional immune infiltrates and ectopic B cell follicles in autopsy multiple sclerosis brains. Variations of CD161 expression on CD8(+) T cells identify a subset of lymphocytes with proinflammatory characteristics that have not been previously reported in multiple sclerosis and are likely to contribute to disease immunopathology.

Prior to constructing a library of yeast artificial chromosomes (YACs) containing very large human DNA fragments, we performed a series of preliminary experiments aimed at developing a suitable protocol. We found an inverse relationship between YAC insert size and transformation efficiency. Evidence of occasional rearrangement within YAC inserts was found resulting in clonally stable internal deletions or clonally unstable size variations. A protocol was developed for preparative electrophoretic enrichment of high molecular mass human DNA fragments from partial restriction digests and ligation with the YAC vector in agarose. A YAC library has been constructed from large fragments of DNA from an Epstein-Barr virus-transformed human lymphoblastoid cell line. The library presently contains 50,000 clones, 95% of which are greater than 250 kilobase pairs in size. The mean YAC size of the library, calculated from 132 randomly isolated clones, is 430 kilobase pairs. The library thus contains the equivalent of approximately seven haploid human genomes.To physically map genomes of complex organisms a number of techniques have been developed. These techniques include pulsed-field gel electrophoresis (1, 2), chromosome jumping (3), and cloning of large DNA fragments in yeast artificial chromosomes (YACs) (4,5). Physical mapping, isolation of large genes plus all regulatory sites, and description of long-range genomic organization will depend on isolating and analyzing fragments of several hundred kilobase pairs of contiguous DNA. Intuitively appealing, the YAC system must be rigorously examined to understand cloning efficiency as a function of size and the extent to which the clones faithfully represent their genomic origin. Until recently (6), all YAC experiments had been carried out with DNA purified and manipulated in solution. In these early experiments one major problem has been small mean size of the YACs. This is probably due to extended handling ofDNA in solution. As can1-100 ura3-1 leul gall 4+ SUQS), W839-1C (a ade2-1 can1-100 or canl-J00,x his3-11,JS leu2-3,112 trpl-1 ura3-1 radl ::LEU2+ radS2-8::TRPI +), Y90 (a ura3-52 lys2-801 ade2-101 trpl-901), Y93 (a ura3-52 lys2-801 ade2-101 trpl-901 his3-A200), and YNN281 (a trpl-A his3-A200 ura3-52 lys2-801 ade2-1 gallb). Media. All yeast cultures were grown on either complete medium (YPD) or selective medium lacking uracil or uracil and tryptophan. These media have been described by Sherman et al. (10 4256The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.