Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors.

The assembly of newly synthesized DNA into chromatin is essential for normal growth, development, and differentiation. To gain a better understanding of the assembly of chromatin during DNA synthesis, we identified, cloned, and characterized the 180- and 105-kDa polypeptides of Drosophila chromatin assembly factor 1 (dCAF-1). The purified recombinant p180+p105+p55 dCAF-1 complex is active for DNA replication-coupled chromatin assembly. Furthermore, we have established that the putative 75-kDa polypeptide of dCAF-1 is a C-terminally truncated form of p105 that does not coexist in dCAF-1 complexes containing the p105 subunit. The analysis of native and recombinant dCAF-1 revealed an interaction between dCAF-1 and the Drosophila anti-silencing function 1 (dASF1) component of replication-coupling assembly factor (RCAF). The binding of dASF1 to dCAF-1 is mediated through the p105 subunit of dCAF-1. Consistent with the interaction between dCAF-1 p105 and dASF1 in vitro, we observed that dASF1 and dCAF-1 p105 colocalized in vivo in Drosophila polytene chromosomes. This interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin.

Circulating immune complex levels in patients with gestational trophoblastic neoplasia.

The clinical course, human chorionic gonadotropin (HCG) levels, and serial circulating immune complex (CIC) levels in 21 patients with gestational trophoblastic neoplasia (GTN) were correlated for the evaluation of the relationship between CIC levels and trophoblastic tumor burden. CIC levels were normal in 18 of 21 patients at the time of presentation, and 2 of 3 patients who presented with elevated CIC levels had significant comorbid disease (toxemia and hepatitis). Nine patients were followed into gonadotropin remission, and all 9 developed an increase in CIC levels at the time of remission. It was concluded that CIC, at least as measured by two antigen-nonspecific techniques, is generally not elevated at initial presentation in the patient with GTN; this lack of an elevation is probably due to marked tumor antigen excess. Thus the in vivo importance of CIC as a "blocker" of host antitumor response at this stage is doubtful. After effective treatment as HCG levels return to normal, the demonstrated elevation in serial levels of CIC may reflect a return of adequate host immune response at a time of minimal tumor burden.

Serial circulating immune complex levels and mitogen responses during progressive tumor growth in WF rats.

Inbred male WF rats were given im injections of one of two antigenically and histologically distinct syngeneic tumor isografts, adenocarcinoma DMH-W 163 or spontaneous renal cell carcinoma SPK. Serum and peripheral blood lymphocytes were harvested from tumor-bearing and normal age-matched controls before and after isograft challenge at weekly intervals. Serial circulating immune complex (CIC) levels were quantitated by polyethylene glycol (PEG) insolubilization. T-cell mitogen responses to phytohemagglutinin (PHA) and concanavalin A (Con A) were followed serially. Tumor growth was measured at least weekly. PEG-CIC values rose early after tumor injection, increased with tumor growth, and declined in some animals just before death. Mitogen response to PHA was significantly decreased in isografted tumor-bearing rats, particularly at later stages of tumor development, compared to normal uninoculated controls. Responses to Con A were variable, and suppression was not always seen in tumor bearers. In animals that did not have progressive tumor growth after isograft injection, PEG-CIC levels did not change and responses to PHA were not suppressed. Patterns of CIC change and responses to PHA were not affected by differences in tumor histology or growth rates. Thus serial CIC levels measured by the PEG assay correlate with tumor growth and precede nonspecific suppression of T-cell mitogenic response in these animal tumor models.

Genetic analysis of mutations at the Glued locus and interacting loci in Drosophila melanogaster.

A genetic analysis of the dominant mutation Glued that perturbs the development of the normal axonal architecture of the fly's visual system was undertaken. Ten new alleles at this locus were identified and characterized. Two complementation groups that were identified failed to complement the original allele, suggesting that it is a double mutant or that it resides at a complex locus. Several of the new alleles display visual-system abnormalities similar to those of the original mutation. Seven of the eight members of one complementation group are embryonic/early larval lethals, like the original mutation. The other allele in this group is temperature sensitive. Homozygous mutant adults exhibit a temperature-sensitive female sterile phenotype. Unsuccessful attempts to recover genetic mosaics carrying clones of cells homozygous for some of these mutations revealed that they are either essential for the viability of individual cells or that they affect some other fundamental cellular function, such as mitosis or the ability to participate in tissue level organization, which prevents them from being recovered in adult mosaics. This also indicates that these mutations do not specifically affect neural cells. A number of X-ray- and EMS-induced partial and complete phenotypic "revertants" of the original allele have also been isolated as material for comparative analysis of visual system development. All "revertants" that alter the abnormal eye phenotype towards the wild type have similar impact on the organization of the optic lobe.

The extra sex combs protein is highly conserved between Drosophila virilis and Drosophila melanogaster.

Extra sex combs (esc) is one of the Polycomb Group genes, whose products are required for long term maintenance of the spatially restricted domains of homeotic gene expression initially established by the products of the segmentation genes. We recently showed that the esc protein contains five copies of the WD motif, which in other proteins has been directly implicated in protein-protein interactions. Mutations affecting the WD repeats of the esc protein indicate that they are essential for its function as a repressor of the homeotic genes. We proposed that they may mediate interactions between esc and other Polycomb Group proteins, recruiting them to their target genes, perhaps by additional interactions with transiently expressed repressors such as hunchback. To further investigate the functional importance of the WD motifs and identify other functionally important regions of the esc protein, we have begun to determine its evolutionary conservation by characterizing the esc gene from Drosophila virilis, a distantly related Drosophila species. We show that the esc protein is highly conserved between these species, particularly its WD motifs. Their high degree of conservation, particularly at positions which are not conserved in the WD consensus derived from alignment of all known WD motifs, suggests that each of the WD repeats in the esc protein is functionally specialized and that this specialization has been highly conserved during evolution. Its highly charged N-terminus exhibits the greatest divergence, but even these differences are conservative of its predicted physical properties. These observations suggest that the esc protein is functionally compact, nearly every residue making an important contribution to its function.

The Drosophila extra sex combs protein contains WD motifs essential for its function as a repressor of homeotic genes.

Extra sex combs is a member of the Polycomb Group genes, whose products are required for stable long term transcriptional repression of the homeotic genes of the Bithorax and Antennapedia complexes. The Pc-G proteins are required to maintain the spatially restricted domains of homeotic gene expression established by the transiently expressed repressors, e.g., hunchback, but are not required for the functioning of these early repressors. This implies two distinct modes of repression and raises the question: how does the transition from early transient repression to stable Pc-G-mediated repression occur? While other Pc-G proteins are required continuously throughout development, the esc RNA is only present transiently in early embryos, suggesting that esc may play a role in mediating this transition to stable long term Pc-G-mediated repression. The predicted esc protein contains multiple copies of the WD motif, found in G-protein beta subunits as well as non-G proteins involved in diverse cellular functions, including transcriptional repression. The sequence alterations of a number of esc mutations cause amino acid substitutions within the WD repeats, identifying them as essential for the function of the esc protein as a repressor of homeotic gene expression. Other WD proteins are components of reversible macromolecular assemblies and the WD motif has recently been directly implicated in mediating interactions with other proteins in such complexes. We propose that the esc protein is similarly involved in the initial recruitment of Pc-G repressors to the homeotic genes to establish their stable long term repression.

Molecular characterization of the trithorax gene, a positive regulator of homeotic gene expression in Drosophila.

The Drosophila gene trithorax (trx) is required for the normal expression of a number of the homeotic genes in the bithorax complex (BX-C) and the Antennapedia complex (ANT-C). Flies homozygous for trx mutations exhibit segmental identity transformations similar to those caused by loss-of-function mutations in the homeotic genes Sex combs reduced (Scr), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B). We present a molecular characterization of the trx locus and show that it is necessary for normal levels of Antennapedia (Antp), Ubx, and abd-A protein accumulation. Interestingly, the loss of trx function differentially affects the expression these proteins; Ubx protein levels are greatly reduced, abd-A protein levels are reduced to a lesser extent, and Antp protein levels are only slightly reduced. P-element mediated transformation using 34 kb of genomic DNA containing the 25 kb trx transcription unit identifies all sequences necessary for normal trx function and limits the 5' and 3' flanking sequences that could be used in a regulatory capacity to relatively small regions. The primary transcription unit is differentially spliced to produce two large transcripts of 12 and 15 kb that have different developmental profiles.

Trithorax is required to maintain engrailed expression in a subset of engrailed-expressing cells.

We show that maintenance but not initiation of engrailed (en) gene expression in the Drosophila embryo requires trithorax (trx), which is also required to maintain stable long-term expression of the homeotic genes throughout the development. Like the homeotic genes, en expression is dependent on trx in only a subset of embryonic cells normally expressing en, including specific cells in the nervous system and the dorsal fat body cells surrounding the gonad. Loss of en expression in the dorsal fat body is correlated with the sterility of en females which also carry trx mutations. In addition, trx is required for normal en expression in the posterior compartment of the developing wing, reflected in enhancement of en phenotypes in en adults which also carry trx mutations. trx appears to be dispensable for maintenance of en expression in other embryonic cells. The trx protein binds to the region of the polytene chromosomes which contains the en gene, suggesting that trx regulates en expression directly by binding to the en regulatory region.

The Drosophila trithorax proteins contain a novel variant of the nuclear receptor type DNA binding domain and an ancient conserved motif found in other chromosomal proteins.

The products of the trithorax gene are required to stably maintain homeotic gene expression patterns established during embryo-genesis by the action of the transiently expressed segmentation genes. We have determined the intron/exon structure of the trx gene and the large alternatively spliced trx RNAs, which are capable of encoding only two protein isoforms. These very large trx proteins differ only in a long Ser- and Gly-rich N-terminal extension, encoded by exon II, which is present only in the larger trx isoform. We have identified a novel variant of the highly conserved nuclear receptor type of DNA binding domain. We have found that the previously identified Cys-rich central region contains multiple novel zinc finger motifs which are also present in the Polycomb-like protein and RBP2, a retinoblastoma binding protein. The trx proteins terminate with another novel conserved domain which we have identified in proteins from three kingdoms, including plants and fungi, indicating that has an ancient origin. Many of these proteins are chromosomally associated, suggesting that this domain may be involved in interactions between trx and other highly conserved components of chromatin involved in transcription regulation. The sequence alterations of trx mutations identify the highly conserved regions of trx as critical for the function of these large proteins. We show that zygotically expressed trx RNAs encoding the larger protein isoform are initially expressed in a spatially restricted pattern which overlaps the expression domains of the BX-C genes Ubx, abd-A and Abd-B. This pattern is transient and evolves into a broader expression domain encompassing the entire germ band during the extended germ band stage.

Suppurative inflammation of vas deferens: an unusual groin mass.

We report a case of suppurative vasitis that presented as an inguinal mass in a young man. Inflammation of the vas deferens may occur as a manifestation of genital tuberculosis or other genitourinary tract infection. A granulomatous vasitis, vasitis nodosa, is usually associated with previous trauma or surgery. Suppuration is rare. We report a case that represents a rare cause of an inguinal mass.

Application of polyethylene glycol turbidity assay to detection of circulating immune complexes in cancer patients.

A rapid, reproducible immune complex screening assay was used to quantitate levels of circulating immune complexes in the sera of normal subjects, patients with documented increases in immune complexes from rheumatoid arthritis, patients with clinically or microbiologically documented infections, and patients with cancer. Although wide variations in individual values within the groups were noted and the concurrent elevation of polyethylene glycol-circulating immune complex levels by infection was documented as expected, significant differences were found in the values in patients with cancer compared with those in normal subjects. The overall clinical application of polyethylene glycol-circulating immune complex screening is discussed and current application of screening of serial sera samples from individual patients for correlation with measurable tumor volume is proposed.

Biliary surgery for benign disease a study of 500 consecutive operations.

An analysis of 500 consecutive operations on the biliary tract for benign disease, performed over a 5 year period is presented. Twenty-two per cent of patients had common duct exploration, and the incidence of choledochotomy increased with advancing age. The overall incidence of choledocholithiasis was 15.2% and calculi in the common duct were found more frequently in the elderly. The use of operative cholangiography increased over the 5 year study period, but was not associated with any change in the incidence of common bile duct exploration or choledocholithiasis. Four patients had re-exploration for retained calculus (0.8%). The overall operative mortality was 0.6%.

Trithorax regulates multiple homeotic genes in the bithorax and Antennapedia complexes and exerts different tissue-specific, parasegment-specific and promoter-specific effects on each.

The trithorax (trx) gene is required for normal development of the body plan in Drosophila embryos and adults. Mutations in trx cause homeotic transformations throughout the body. Genetic studies suggest that trx encodes a positive regulatory factor required throughout development for normal expression of multiple homeotic genes of the bithorax and Antennapedia complexes (BX-C and ANT-C). To determine how trx influences homeotic gene expression, we examined the expression of the BX-C genes Ultrabithorax, abdominal-A, Abdominal-B and the ANT-C genes Antennapedia, Sex combs reduced and Deformed in trx embryos. We show that trx does indeed exert its effects by positively regulating homeotic gene expression and that its effects on expression of individual homeotic genes are complex: each of the BX-C and ANT-C genes examined exhibits different tissue-specific, parasegment-specific and promoter-specific reductions in their expression. This implies that each of these genes have different requirements for trx in different spatial contexts in order to achieve normal expression levels, presumably depending on the promoters involved and the other regulatory factors bound at each of their multiple tissue- and parasegment-specific cis-regulatory sites in different regions of the embryo. These results also imply that those components of homeotic gene expression patterns for which trx is dispensable, require other factors, possibly those encoded by other trithorax-like genes.

Analysis of visual system development in Drosophila melanogaster: mutations at the Glued locus.

We have analyzed several aspects of the development of flies carrying mutations at the Glued locus. Optic lobe abnormalities in individuals heterozygous for the original Glued allele were previously shown to result from an action of this mutation in the retinula cells. We have estimated when the functioning of this gene or its product is required for normal visual system development by using genetic mosaicism induced by somatic recombination and temperature shifts of a temperature-sensitive mutation at this locus. Both methods point to a period in the mid-third instar, suggesting that early events in the formation of ommatidia and/or late events in the program of retinal cells are affected. Application of a new histological stain for developing axons indicates that individuals heterozygous for Glued exhibit abnormalities in the retinula fiber projection by the late third instar. Thus, the adult phenotype is not solely the result of later cellular degeneration or rearrangement. Beneath M+ Gl+ clones which encompass the entire eye were found optic lobe abnormalities with features not seen in either other mosaics or Gl heterozygotes. The possibility that these abnormalities result from temporal asynchrony in the development of eye and and optic lobe in these individuals is discussed and the results of attempts to test this hypothesis are presented.

The Drosophila trithorax protein binds to specific chromosomal sites and is co-localized with Polycomb at many sites.

trithorax is required to stably maintain homeotic gene expression patterns established during embryogenesis by the action of the transiently expressed products of the segmentation genes. The large trithorax proteins contain a number of highly conserved novel motifs, some of which have been hypothesized to interact directly with specific DNA sequences in their target genes. Using antibodies directed against trithorax proteins, we show that they are bound to 63 specific sites on the polytene chromosomes of the larval salivary gland. trithorax binding is detected at the sites of its known targets, the Bithorax and Antennapedia complexes, despite the transcriptionally repressed state of these loci in the salivary gland. A temperature-sensitive trithorax mutation greatly reduces the number of binding sites. Simultaneous localization of trithorax and Polycomb indicates that many of their chromosomal binding sites coincide. We localized one trithorax binding site within a portion of the large 5' regulatory region of the Ubx gene, to an interval which also contains binding sites for Polycomb group proteins. These results suggest that trithorax exerts its effects by binding directly or indirectly to specific DNA sequences in its target genes. Co-localization with Polycomb also suggests that interactions between these activators and repressors of the homeotic genes may be a significant feature of their mode of action.

Paratesticular leiomyosarcoma: an ultrastructural study.

We report a case of paratesticular leiomyosarcoma in an 84-year-old man. The tumor was firm and nodular, and attached to the upper pole of the left testis. Light microscopy showed a sarcoma with numerous mitoses. On electron microscopy tumor cells contained 7 nm. microfilaments, with regular dense bodies, micropinocytotic vesicles and the other hallmarks of smooth muscle differentiation, in addition to prominent pools of glycogen. This case illustrates the value of electron microscopy in the investigation of unusual urological tumors.