In vivo ionizing irradiations produce deletions in the hprt gene of human T-lymphocytes

The hprt T-lymphocyte cloning assay, which detects mutations occurring in vivo in humans, has been used to examine mutants induced in patients receiving radioimmunoglobulin therapy (RIT) for cancer. Samples from 13 patients before treatment (controls) and 15 samples from 12 patients after treatment were studied for both mutant frequencies and molecular changes in the hprt mutant T-cell clones. Patients were studied up to 48 months after treatment. Post-RIT patients showed increased mutant frequencies as compared to pre-treatment values. T-cell receptor (TCR) gene analysis of mutant T-cell clones demonstrated that 84% arose independently, both pre- and post-treatment, which is the same proportion as seen in normal individuals. However, several individuals did show large sets of mutants with the same TCR gene rearrangement patterns. Molecular analysis of mutants demonstrated a greater proportion of mutations with hprt gene changes on Southern blots after RIT treatment than before (40% versus 20%). RIT increases the proportion of mutations with total rather than partial gene deletions or other gross structural changes compared to normal individuals or pre-treatment patients. These studies are defining the spectrum for radiation-induced hprt gene mutations in vivo in human T-lymphocytes.     

Mechanisms of Mutagenesis by a Bulky DNA Lesion at the Guanine N7 Position

The RpII215 locus encodes the large subunit of RNA polymerase II (polII). Three of 22 RpII215 alleles cause a synergistic enhancement of the mutant phenotype elicited by mutations in the Ultrabithorax (Ubx) locus. We have recovered and analyzed three new mutations that suppress this enhancement. All three mutations map to the RpII215 locus. In addition to suppressing the Ubx enhancement of other RpII215 alleles, two of the new mutations, JH1 and WJK2, themselves enhance Ubx. RpII215 alleles can be placed into three classes based on their ability to enhance Ubx. Class I alleles, including Ubl, C4, C11, JH1, and WJK2, enhance Ubx when heterozygous with class II alleles, which include wild-type RpII215. Class III alleles, which include amorphic alleles, do not enhance Ubx. The third new mutation, WJK1, is a conditional amorphic allele, which behaves like a class III allele at 29 degrees but like a class II allele at 19 degrees. Another mutant phenotype is caused by certain RpII215 alleles, including all class I alleles. This phenotype is a synergistic enhancement of a mutant phenotype elicited by mutations at the Delta (Dl) locus. Unlike the enhancement of Ubx, the enhancement of Dl is not dependent upon antagonistic interactions between different classes of RpII215 alleles.     

Phosphorus in sediments — speciation and analysis

Characterization of sediment phosphorus is commonly based on sequential chemical extractions, in which phosphorus is supposed to be selectively removed from different compounds in the sediments. The first extraction schemes were designed to quantify discrete chemical or mineralogical compounds. As extraction schemes have been tested on different sediments, several systematic errors have been detected and the schemes have been modified and simplified accordingly. Other chemical extractions or treatments have attempted to determine phosphorus bound to particles with a certain strength or binding energy, the purpose being to determine the labile, loosely bound, exchangeable, mobile or algal-available fraction of sediment phosphorus. All extraction procedures yield operationally defined fractions and cannot be used for identification of discrete phosphorus compounds. The many methodological modifications make it necessary to be cautious when comparing results from the literature in this field.     

Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes

T-cell receptor (Ti) gene restriction fragment patterns (RFPs) were determined by Southern blots of genomic DNA obtained from T-lymphocyte colonies isolated from a single normal individual. 4 wild-type colonies and 11 in vivo derived 6-thioguanine-resistant mutant colonies with previously characterized hprt gene structural alterations were studied. Among the hprt mutants, 10 of the 11 showed unique Ti RFPs indicating their origins in different in vivo progenitors. Unique Ti RFPs were also seen among the wild-type T-cell colonies. One, however, shared its Ti RFP with a mutant. These results suggest that mutation in vivo of the hprt gene in human T-lymphocytes occurred after thymic maturation and that the 11 recovered hprt mutants probably resulted from 11 independent mutational events.     

Antibody-nucleic acid interactions. Monoclonal antibodies define different antigenic domains in 2',5'-oligoadenylates

To define the epitopes involved in binding anti-oligonucleotide antibodies, several hybridomas producing monoclonal antibodies directed against 2',5'-oligoadenylate were established. A solid-phase enzyme-linked immunoassay that employed microtiter wells coated with Ficoll-2',5'-oligoadenylate conjugates proved useful in screening and characterizing hybridoma supernatants. Control experiments demonstrated that the conjugates were irreversibly adsorbed to polystyrene wells under the conditions employed in the assay. Reactivity of monoclonal antibodies with numerous analogues of 2',5'-oligoadenylate was measured by using a competition assay. Several monoclonal antibodies originating from different mice immunized with the same or different immunogens possessed distinctive fine specificities. At least one 2',5'-phosphodiester bond was important in forming each epitope, suggesting that the ribose phosphate backbone is a critical element in defining an antigenic domain of an oligonucleotide. The purine bases were also important, and modification of the bases had varied effects on the extent of antibody recognition. The length of the oligonucleotide and the nature of the termini were also of some importance. In several instances the modification created by linkage of 2',5'-oligoadenylate to carrier protein also contributed to the determinant. The monoclonal antibody most specific for 2',5'-oligoadenylates was relatively insensitive to ionic strength. In contrast, a monoclonal antibody with a 2',5'-oligopurine specificity appeared to bind 2',5'-oligoadenylate through one ion pair, whereas the binding of a monoclonal antibody with a low degree of base specificity appeared to bind through two ion pairs. The results demonstrated that 2',5'-linked oligoadenylate-protein complexes possess at least three distinct oligonucleotide-related antigenic surfaces that can be recognized with high apparent affinity by monoclonal antibodies. A model for the three epitopes is presented.     

Genotype-specific soluble auxin-binding in etiolated pea epicotyls

Using different independent procedures for assaying soluble auxin-binding in etiolated pea epicotyls, wo could prove the reliability of the (XH₄)₂SO₄-pelleting assay both for crude cytosols as well as for specific protein fractions obtained after chromatofocusing. Three distinct genotypes (two parent lines, one tall recombinant) investigated so far exhibit characteristic differences with respect to soluble auxin-binding kinetics in their cytosols.     

Lupus prone (SWR x NZB)F1 mice produce potentially nephritogenic autoantibodies inherited from the normal SWR parent

The incidence of nephritis in autoimmune NZB mice is low, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. To define the contribution of the normal SWR strain to the development of nephritis, we analyzed 65 monoclonal anti-DNA autoantibodies derived from SNF1 mice and compared them with those obtained from the NZB parent. The majority of the SNF1-derived anti-DNA antibodies were IgG and cationic in charge. By contrast, 77% of the NZB-derived antibodies were IgM. Moreover, all three NZB-derived IgG anti-DNA antibodies were anionic. The cationic property of the SNF1-derived IgG autoantibodies was not restricted to any particular antigenic specificity pattern or IgG subclass, nor was there a preference for the allotype of either parent. However, we identified a set of highly cationic (pI at 8.2 to 8.8 pH) IgG2b anti-DNA antibodies from SNF1 hybrids that had the SWR allotype. Isoelectric focusing of intact antibodies and isolated heavy and light chains showed that the highly cationic charge of these antibodies was determined by the variable regions of their heavy chains. Because IgG anti-DNA antibodies with cationic charge are especially pathogenic, those antibodies bearing the allotype of the normal SWR parent may account for the high incidence of severe nephritis in the F1 hybrids. The results indicate that pathogenic autoantibodies, which are encoded by genes of the nonautoimmune SWR parent, are expressed in the SNF1 mice due to some cellular and genetic regulatory influence of the NZB parent.     

Interspecific crosses inHordeum (Poaceae)

A crossing programme including 30 species and 40 cytotypes within the genusHordeum was undertaken. Viable hybrids were obtained in 302 combinations, 15 of which were intraspecific. Differences in seed set and in germination were observed in crosses between different groups of species. Obtaining crosses between different taxonomic groups was generally more difficult when diploid material was used. Some species, e.g.,H. lechleri, H. jubatum, andH. brachyantherum showed a higher crossability than others. The chromosome numbers of the hybrids were usually those expected from the parental numbers but aneuploid series around the expected numbers were rather frequent. Three cases of unreduced gametes were found. Selective chromosome elimination was restricted to combinations including eitherH. vulgare orH. bulbosum.—Despite a very diverse morphology, all South American diploid species together with the two North American diploidsH. intercedens andH. pusillum appear to be closely related. The hexaploid American speciesH. procerum, H. lechleri, andH. arizonicum are also related. The two North American tetraploid speciesH. jubatum andH. brachyantherum sometimes form semifertile hybrids. The Asiatic speciesH. roshevitzii appears to be related to both North and South American taxa.     

Thymic epithelial genotype influences the production of recombinant leukemogenic retroviruses in mice

By using T1 oligonucleotide fingerprinting and mapping techniques, we analyzed the genomic structure of retroviruses produced by thymocytes and splenocytes of reciprocal bone marrow-and thymus-grafted chimeras. We found that the genetic factor(s) derived from NZB mice that suppresses the development of thymic leukemia in (AKR X NZB)F1 mice also prevents the formation of recombinant leukemogenic viruses and the expression of preleukemic changes in the (AKR X NZB)F1 thymocytes. The NZB mouse gene or genes appeared to exert this suppressive effect by acting on the thymic reticuloepithelial cells and not on the thymic lymphocytes of (AKR X NZB)F1 hybrids. Prospective studies with thymic epithelial grafts from young mice showed that the AKR thymic epithelium could mediate the formation and expression of leukemogenic recombinant viruses and preleukemic changes in thymocytes that lead to the development of thymic leukemia, whereas the (AKR X NZB)F1 thymic epithelium was deficient in this regard. Our results also confirmed a previous observation that during in vivo generation of recombinant leukemogenic viruses, the acquisition of polytropic virus-related sequences in the 3' portion of the p15E gene and the U3 region and in the 5' part of the gp70 gene can occur independently.     

CHROMOSOME MICROMANIPULATION : III. Spindle Fiber Tension and the Reorientation of Mal-Oriented Chromosomes

Kinetochore reorientation is the critical process ensuring normal chromosome distribution. Reorientation has been studied in living grasshopper spermatocytes, in which bivalents with both chromosomes oriented to the same pole (unipolar orientation) occur but are unstable: sooner or later one chromosome reorients, the stable, bipolar orientation results, and normal anaphase segregation to opposite poles follows. One possible source of stability in bipolar orientations is the normal spindle forces toward opposite poles, which slightly stretch the bivalent. This tension is lacking in unipolar orientations because all the chromosomal spindle fibers and spindle forces are directed toward one pole. The possible role of tension has been tested directly by micromanipulation of bivalents in unipolar orientation to artificially create the missing tension. Without exception, such bivalents never reorient before the tension is released; a total time "under tension" of over 5 hr has been accumulated in experiments on eight bivalents in eight cells. In control experiments these same bivalents reoriented from a unipolar orientation within 16 min, on the average, in the absence of tension. Controlled reorientation and chromosome segregation can be explained from the results of these and related experiments.