c or reduced phenazine methosulfate coupled with the reduction of ubiquinone. We wish to report now that chromatophores from aerobically grown R. spheroides, strain Ga, sensitize these reactions efficiently (with quantum requirements of a few quanta per electron transfer), whereas chromatophores from the nonphotosynthetic mutant strain PM-8 are entirely unable to drive these photochemical processes. The failure of strain PM-8 to catalyze the photooxidation of reduced phenazine methosulfate is the more remarkable because this reaction is sensitized by purified bacteriochlorophyll in vitro. These findings show that the major component of bacteriochlorophyll is inert with respect to the foregoing light-induced activities, and that a special kind of reaction center is needed for the photochemistry that leads to photosynthesis. The results of experiments with exogenous reagents will be published in detail elsewhere. We are indebted to Dr. W. S. Zaugg for making available both the chemicals and the methodology for studying the photochemical electron transfer reactions of cytochrome, phenazine methosulfate, and ubiquinone. * Contribution no. 157 from the Charles F. Kettering Research Laboratory.

1. Blood group substances have been prepared from human saliva, stomach, and amniotic fluid from individuals of blood group A1 and A2. Several of the saliva samples were obtained from individuals shown to be heterozygous, A1O. 2. The purified blood group A substances from human sources were similar in nitrogen, glucosamine, reducing sugar, and acetyl content. The A1 and A2 substances differed in optical rotation. All of the human A samples were levorotatory while those from hog stomach were dextrorotatory. 3. By two immunochemical criteria the various human preparations could be shown to fall into distinct groups, with respect to purity. The best products showed maximal activity and almost all of their glucosamine was specifically precipitable by anti-A. These samples of human A substance were only about one-half as effective in precipitating antibody to hog A substance formed in man as was homologous hog A substance although the same total amount of antibody was precipitable by excess of either antigen. 4. Human blood group A1 substance was found to be antigenic in individuals of blood groups B and O but was not as good an antigen as hog A substance.

A B S T R A C T Renal tissues from two groups of patients were studied with fluorescein-labeled (Fl-) antibodies (Abs) to immunoglobulins, complement, and antibodies prepared in rabbits against BSA conjugate of 5-methyluridine (T) and cytidine (C), the latter two of which react specifically with denatured DNA. The first group consisted of 13 SLE patients, and the second consisted of 53 patients with non-SLE nephropathies. The data obtained from the two groups of patients were used for comparison, and they showed the following:(a) Fl-Abs to immunoglobulins and complement were bound in the glomeruli of tissues from all patients with active SLE glomerulonephritis characterized by deposits of foreign material in glomerular capillary walls (GCW). The fluorescent pattern was granular, corresponding to the distribution of the glomerular deposits, as seen by electron microscopy. Fl-Abs reactive with thymine and cytosine were bound in the GCW of eight of the nine patients with active SLE glomerulonephritis and showed the same granular distribution. The capacity of these latter Fl-Abs to stain the GCW was removed by absorption with the homologous antigen or denatured DNA.(b) Fl-Abs to immunoglobulins, complement, and pyrimidine bases of DNA did not react with the GCW of two SLE patients without clinical and histologic evidence of glomerulonephritis or with the sclerotic glomeruli of two uremic patients with chronic "burned out" lupus nephritis.(c) The glomeruli of 47 of the 53 patients with other nephropathies bound Fl-Abs to immunoglobulins and

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Substances that have the properties of antibodies and react with DNA from various sources are frequently found in the sera of patients with systemic lupus erythematosus." 2 They appear to be formed spontaneously, perhaps as autoantibodies, but the stimulus for their production is not known.There have been many attempts to induce the formation of antibodies with specificity towards DNA,3-5 but those which react with purified DNA have been conclusively demonstrated in only one instance.6 In this example, the antigen, a partially denatured DNA from T4 bacteriophage, was unusual in that it contained a glucosylated pyrimidine, and the antibody produced was found to be specific towards this antigenic component.' DNA from sources other than T-even phages did not react with this antibody.Although antibodies specific for a variety of haptens have been obtained following the classical technic of Landsteiner,8 no record has been found of the use of nucleic acid components for such studies. Accordingly, purines and pyrimidines were covalently linked to protein carriers to determine whether they might stimulate the formation of antibodies. Bovine serum albumin conjugates containing the [6-purinyl]-sulfonyl, orotic acid, and [6-purinyl]-f3-alanyl moieties were prepared, but antibodies to these antigens gave equivocal results when examined for purine or pyrimidine specificity.9 This report describes the coupling of 6-trichloromethylpurine'0 to serum albumin, and the use of the resulting conjugate as an antigen to induce the formation of antibodies which exhibit a purine specificity and react with DNA.Materials and Methods.-Purinoyl-protein conjugates: These substances were synthesized by a method which was derived from a previously described technic1' for the preparation of aroylprotein conjugates. A 'chilled solution containing 750 mg bovine serum albumin (BSA; Pentex Fraction V) and 170 mg 6-trichloromethylpurine in 70 ml of 4% tetrahydrofuran-water was stirred at room temperature for 3 hr. The pH was maintained throughout at 10-10.5 by the addition of 0.1 N NaOH and the solution was dialyzed overnight against running tap water. Upon acidification to pH 4.5 with 0.1 N HCl, the conjugated protein precipitated. It was redissolved in 25 ml of 0.15 M NaHCO3, dialyzed against running water, and lyophilized. The product (Pur-BSA) thus obtained dissolved readily in water or buffer solutions and migrated in agar gel electrophoresis on a microscope slide as a single component toward the anode at pH 8.2 more rapidly than did unconjugated BSA. From spectrophotometric measurements, Pur-BSA was estimated to contain approximately 24 purine groups per mole of BSA (molecular weight, 67,000).12 In the same way, the conjugate (Pur-HSA) with human serum albumin (HSA, Pentex Fraction V) was prepared. It contained about 27 moles of purine per mole. Spectral properties of these derivatives determined in a Cary spectrophotometer in 1 cm cells are given in Table 1.Immunochemical procedures: Rabbits were immunized by injection, into the foot-pads,'3 ...

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Human metaphase chromosomes were photooxidized in the presence of methylene blue, a process that destroys guanine residues in DNA. Indirect immunofluorescence showed that such chromosomes reacted with a cytosine-specific antibody revealing a consistent fluorescent banding pattern by which each chromosome could be identified. The observed fluorescent patterns were the reverse of those produced in formamide-denatured chromosomes treated with an antibody specific for adenine and of the patterns obtained with quinacrine and with Giemsa staining by the G-banding techniques. The patterns were identical to Giemsa R-banding patterns. The chromosome banding patterns, therefore, appeared to reflect DNA base composition, indicating the feasibility of a combined chemical-immunochemical investigation of the chemical organization of chromosomes.It is possible to obtain anti-nucleoside antibodies (1) that are specific for the purine or pyrimidine determinant group of the immunizing antigen. By complement fixation (2) and radioimmunoassay (3), they show no crossreactions with any of the other bases present in nucleic acids. These antibodies react with single-stranded, denatured, or partially denatured DNA, but not with native DNA. They combine with fixed metaphase chromosomes, but only if the chromosomes are partially denatured (4). Using indirect immunofluorescence procedures, Dev et al. (5) found that anti-adenosine (anti-A) produced a characteristic pattern of light and dark bands along human metaphase chromosomes that had been treated for 1 hr at 650 with 95% formamide in the presence of 0.25% formaldehyde. The pattern obtained corresponded closely with those produced by quinacrine or by the Giemsa Gbanding techniques (6-8), a finding consistent with the evidence that enhanced fluorescence of quinacrine occurred in AT-rich regions of DNA (9-11). The results suggest, moreover, that the specificity of anti-nucleoside antibodies observed in solution is carried over to the chromosome system.If anti-nucleoside antisera are indeed as specific in chromosomal preparations as they are in solution, it should be possible to map chromosomes chemically by use of various chemical and immunochemical procedures in combination. The more selective the chemical procedure, the more information one should be able to obtain with anti-nucleoside antibodies. Simon and Van Vunakis (12) have shown that guanine residues are selectively destroyed by photooxidation of DNA in the presence of methylene blue. No breakdown of the other Abbreviations: anti-A and anti-C, adenosine-and cytosinespecific antibodies, respectively; PBS, phosphate-buffered saline pH 7.2-7.4. 804DNA bases was observed. Their results were confirmed by Garro et al. (2), who also demonstrated that the destruction of guanine exposed cytosine residues that could be detected by cytosine-specific antibody (anti-C).

Rabbits immunized with bovine serum albumin conjugates of 5-bromouracil, 5-iodouracil, and 6-methyladenosine produced antibodies specific for the bases. These antibodies were used to detect immunochemically 5-bromouracil and 6-methyladenosine in denatured DNA.