Anchoring of rice BAC clones to the rice genetic map in silico.

A wealth of molecular resources have been developed for rice genomics, including dense genetic maps, expressed sequence tags (ESTs), yeast artificial chromosome maps, bacterial artificial chromosome (BAC) libraries and BAC end sequence databases. Integration of genetic and physical maps involves labor-intensive empirical experiments. To accelerate the integration of the bacterial clone resources with the genetic map for the International Rice Genome Sequencing Project, we cleaned and filtered the available EST and BAC end sequences for repetitive sequences and then searched all available rice genetic markers with our filtered databases. We identified 418 genetic markers that aligned with at least one BAC end sequence with >95% sequence identity, providing a set of large insert clones with an average separation of 1 Mb that can serve as nucleation points for the sequencing phase of the International Rice Genome Sequencing Project.

Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases.

Glutathione S-transferases (GSTs) traditionally have been studied in plants and other organisms for their ability to detoxify chemically diverse herbicides and other toxic organic compounds. Anthocyanins are among the few endogenous substrates of plant GSTs that have been identified. The Bronze2 (Bz2) gene encodes a type III GST and performs the last genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the comparable step in the petunia anthocyanin pathway is controlled by the Anthocyanin9 (An9) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST. Bz2 and An9 have evolved independently from distinct types of GSTs, but each is regulated by the conserved transcriptional activators of the anthocyanin pathway. Here, a phylogenetic analysis is presented, with special consideration given to the origin of these genes and their relaxed substrate requirements. In particle bombardment tests, An9 and Bz2 functionally complement both mutants. Among several other GSTs tested, only soybean GmGST26A (previously called GmHsp26A and GH2/4) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been associated with the anthocyanin pathway. Requirements for An9 and Bz2 gene function were investigated by sequencing functional and nonfunctional germinal revertants of an9-T3529, bz2::Ds, and bz2::Mu1.

Diversification, not use, of the immunoglobulin VH gene repertoire is restricted in DiGeorge syndrome.

Immunoglobulin (Ig) genes were isolated from unamplified conventional as well as polymerase chain reaction-generated cDNA libraries constructed from the peripheral blood cells of a patient with complete DiGeorge syndrome. Comparison of the sequences of 36 heavy chain clones to the recently expanded database of human VH genes permitted identification of the germline VH genes that are expressed in this patient as well as placement of 19 of these genes in a partially resolved 0.8-mb region of the human VH locus. The pattern of VH gene use does not resemble the fetal (early) repertoire. However, as in the fetal repertoire, there are a number of cDNAs derived from germline genes that previously have been identified as autoantibodies. Two D mu sequences also were identified, as was another sequence resulting from a unique recombination event linking JH to an unidentified sequence containing a recombination signal sequence-like heptamer. All of the DiGeorge cDNAs are closely related to germline VH genes, showing little or no evidence of somatic mutation. In contrast, comparably selected IgM VH sequences derived from normal adult and age-matched human libraries, and from a second DiGeorge syndrome patient in whom the degree of thymic dysfunction is much less severe, exhibit considerable evidence of somatic mutation. The absence of somatic mutation is consistent with the atypical development of functional antibody responses associated with complete DiGeorge syndrome and implicates a role for T cells in the generation of diversity within the B cell repertoire.

The effect of dithiothreitol on the kinetics of dissociation of dexamethasone from the non-transformed mammary cytosolic glucocorticoid receptor.

Sulfhydryl reducing agents such as dithiothreitol are required for maximum binding of dexamethasone to the mammary cytosolic glucocorticoid receptor, but little is known concerning the effects of dithiothreitol on the kinetics of the binding reaction. In this report we have examined the influence of dithiothreitol on the dissociation kinetics of dexamethasone from the non-transformed glucocorticoid-receptor complex at 0-4 degrees C under various experimental conditions. Without dithiothreitol, the rate of dissociation of dexamethasone remains essentially the same (t1/2 approximately 17 h) regardless of the method chosen to monitor dissociation. With dithiothreitol, however, there is a marked acceleration in the rate of dissociation of receptor-bound dexamethasone when an excess of unlabeled dexamethasone is used to study dissociation (t1/2 approximately 5 h) but not when dissociation is investigated by removal of free labeled dexamethasone by charcoal adsorption (t1/2 approximately 21 h); dithiothreitol also accelerates the observed rate of dissociation when a combination of these methods is used. An acceleration in the rate of receptor-bound dexamethasone is also observed when an excess of the synthetic progestin, R5020, is used in the dissociation assay. The possible reasons and importance underlying these findings have been discussed.

Autoradiographic demonstration of estrogen binding in human breast cancer after in vitro incubation.

This report describes the localization of bound radioactivity as visualized by thaw-mount autoradiography in 35 cases of human breast carcinoma after in vitro incubation with [3H]estradiol. The findings have been compared qualitatively with results of biochemical assays. Twenty-six tumors were considered to be estrogen receptor positive by autoradiographic criteria. Of these, 23 were assayed biochemically; 21 were found to be positive. Specific uptake of radioactivity was observed primarily in neoplastic epithelial cells. The grains were localized mainly over the nuclear region of putative target cells. Within nests of infiltrating carcinoma, positive cells could be identified admixed with negative cells. There were no autoradiographic criteria established for borderline cases. The remaining nine cases were considered negative. In these cases the appearance of tissue exposed only to [3H]estradiol resembled that of tissue incubated with excess unlabeled estradiol. The few grains were randomly scattered with no evidence of nuclear localization. Of these negative cases, two were positive by biochemical assay, four borderline, and three negative. It is concluded that, as assessed in this investigation, estrogen receptor-positive mammary carcinomas may be composed of target and nontarget neoplastic cells. Thaw-mount autoradiography should prove useful in the laboratory investigation of steroid hormone responsiveness of human breast carcinoma.

Autoradiographic demonstration of 3H-estradiol incorporation in benign human mammary lesions.

This study reports the distribution of 3H-estradiol incorporation as assessed by thaw-mount autoradiography after in vitro incubation of fresh tissue obtained from a series of 17 benign human mammary lesions. In four out of nine cases of fibroadenoma examined, putative estrogen target cells were identified in ducts where the specific labeling was confined to some but not all epithelial cells. Elsewhere in these lesions, negative ducts could be found. Similarly, in three out of eight cases of fibrocystic disease specific labeling was seen over epithelial cells in some areas but not others. Histologically identifiable myoepithelial cells were negative as well as the vast majority of stromal cells.

The localization of 3H-estradiol in estrogen receptor-positive human mammary carcinoma as visualized by thaw-mount autoradiography.

Biochemical assays of human mammary carcinomas for estrogen receptors (ER) are of proven clinical usefulness. The reliability of histochemical or immunocytochemical methods for ER localization are less well established, and less is known about the distribution of estrogen binding proteins in breast cancer. In this report we present results of a study of the uptake and retention of 3H-estradiol after in vitro incubation as visualized by thaw-mount autoradiography in five cases of biochemically estrogen receptor-positive breast cancer. These neoplasms appeared to be composed of a heterogeneous population of labeled and unlabeled tumor cells. The number of nuclear grains varied among putative target cells, and the relative percentage of labeled cells differed from area to area. Non-neoplastic mammary ductal epithelium on occasion revealed significant nuclear labeling, but stromal cells, inflammatory cells, and endothelial cells were generally negative. The possible significance of these findings is discussed.

Autoradiographic demonstration of uptake and retention of 3H-estradiol after in vitro incubation.

An in vitro incubation method is described for the demonstration of 3H-estradiol in sections of mouse uterus by thaw-mount autoradiography. The method involves the incubation of tissue sections in 5 nM 3H-estradiol with a subsequent 2 hr chase in medium containing 3.5 g% bovine serum albumin. The distribution of the silver grains observed compares favorably to that seen by others with dry-mount autoradiography after in vivo injection. The labeling is inhibited by excess estradiol or diethylstilbesterol, but not by progesterone or hydrocortisone. Its subcellular distribution appears predominantly nuclear in presumptive target cells. Some regional variability in degree of labeling is present throughout the sections, but is far less marked within a given area. Because the observed labeling has been retained during a 2 hr chase and can be inhibited, it is likely to represent physiologically significant uptake and retention of 3H-estradiol in target cells. Preliminary results with human mammary carcinoma suggest this method may be applicable to the investigation of estrogen target cells in human tissue.

Elastosis in benign sclerosing ductal proliferation of the female breast.

Elastosis, an abundance of elastic tissue, is commonly present in breast carcinoma. However, its diagnostic significance remaines an unsettled issue. This study documents 17 cases of elastosis occurring in a distinctive benign sclerosing ductal lesion of the female breast (Fenoglio and Lattes: Cancer 33: 691-700, 1974). Elastosis was characterized by staining reactions and, in several instances, by elastase digestion and electron microscopy. Yellow streaks and flecks may be apparent grossly and probably reflect the increased elastic tissue. Histologically, the lesion is generally stellate with central sclerosis and marked peripheral intraductal and ductular hyperplasia which is often papillary. Elastosis, which may be marked, is a constant finding and is predominantly periductal in location. It is emphasized that the gross and histologic features of the lesion may mimic carcinoma and that elastosis may be found in benign ductal lesions of the breast.

Distribution of adenosine triphosphatase in infiltrating ductal carcinoma and non-neoplastic breast.

The histochemical reaction for adenosine triphosphatase (ATPase) has previously been used to differentiate myoepithelial from epithelial cells in the breast and to investigate the possible contribution of myoepithelial cells to mammary carcinoma. Discrepancies in published reports prompted this study of ATPase in non-neoplastic breast and infiltrating ductal carcinoma. ATPase was localized mainly on myoepithelial cells of normal breast and was identified with significant frequency on epithelial cells in hyperplastic ducts. Infiltrating ductal carcinomas usually displayed a variable reactivity. In one instance, malignant cells demonstrating mucin production were found to be ATPase-positive. An infiltrating ductal carcinoma of the papillary type with apocrine features was also strongly ATPase-reactive. It is concluded that ATPase is not an exclusive marker of myoepithelial cells and, therefore, data resulting from the use of this enzyme to study the role of the myoepithelium in mammary carcinoma must be interpreted with caution.

Endobronchial plasma cell granuloma (xanthomatous pseudotumor); a light and electron microscopic study.

Plasma cell granuloma (xanthomatous pseudotumor) is polymorphic at light as well as electron microscopic examination. At light microscopy the endobronchial variant of this entity was rich in plasma cells and interwoven, whorl-like, or concentrically arranged spindle cells. Foamy histiocytes and macrophages usually abundant in the intrapulmonary variant were rare. At electron microscopy particles 20 to 50 nm. in size were found at the bronchial mucosal surface but not elsewhere in the lesion. Plasma cells near the bronchial surface contained cytoplasmic fibrils, mitochondria with concentric cristae, and inclusions that bore a close resemblance to adjacent extracellular crystallized hemoglobin. Those in the center of the lesion ordinary round inclusions and none of the other changes. Spindle cells in the interlaced areas were mostly fibroblasts or myofibroblasts, whereas those whorled around capillaries resembled pericytes with basement membranes and nuxes-like intercellular junctions. Year rings like multilayered basal laminae were frequently present between the pericyte-like cells and the endothelial cells of the capillaries. The ultrastructure of plasma cell granuloma, like the histologic and clinical aspects, differs from that of sclerosing hemangioma, pseudolymphoma, and malignant plasma cell tumor affecting the lung.