LARALink: a web application for cytogenetic linkage analysis.

Genomic and expression data have increased dramatically over the last several years. This is primarily due to the completion of the human genome project as well as an upsurge in the use of various high-throughput technologies. Recent attempts to correlate genomic and expression data have stimulated the scientific community to determine how this data can be used within a clinical setting (P Khatri et al., Genomics 2002: 79: 266; LJ van't Veer et al., Nature 2002: 415: 530). LARALink (Loci Analysis for Rearrangements Link) is a database-driven web application that utilizes several public datasets to analyze clinical cytogenetic data to identify candidate genes. LARALink allows UniGene clusters or single-nucleotide polymorphisms (SNPs) to be queried for multiple patients by cytoband, chromosome marker, or base pair. The results can be further refined with the use of an anatomical site, developmental stage, pathology, or cell-type expression filter. Once a set of UniGene clusters (expressed genes) has been identified either for a single patient or for a shared region among multiple patients, the expression-distribution profile, expressed sequence tags (ESTs), or online mendelian inheritance in man (OMIM) entries are displayed. The utility of this tool is shown by its application to both research and clinical medicine. LARALink is a public resource available at: http://www.laralink.bioinformatics.wayne.edu:8080/unigene.

Design and implementation of an introductory course for computer applications in molecular genetics. A case study.

Formal training in computational biology was initiated at Wayne State University in 1990 to meet the needs of the faculty. This was still at a time when the molecular databases and analysis tools could be housed in what is now equivalent to a modern but dated desktop computer. In 1995 the course was expanded to include graduate students to provide these senior students with a foundation in computational biology. This course has armed our students with a requisite set of basic skills that are necessary for a successful career in molecular genetics. It is now an integral component of the graduate program of the Center for Molecular Medicine and Genetics and our experiences in course delivery have been detailed (BioInformatics Methods and Protocols, S. Misener and S. A. Krawetz, eds., Humana Press, Totowa, NJ, 2000.). The course was expanded to a campus-wide unlimited enrollment program for the summer of 2000 to address the needs of our student body. In this review we present our experience with delivering a multidisciplinary campus-wide computational biology course to a new and widely diverse student body.

Web-site-based recruitment for research studies on abdominal aortic and intracranial aneurysms.

Our current understanding on the pathogenesis of abdominal aortic and intracranial aneurysms is limited, but genetic and environmental factors as well as their interactions are likely to play important roles in the development and rupture of aneurysms. To identify genetic factors contributing to these diseases, we are carrying out genome-wide screening studies, which require a large number of patients and family members. Current methods of finding patients who qualify for genetic studies are, however, often costly and ineffective. To improve patient recruitment, a Web site was developed (cmmg.biosci.wayne.edu/ags). The site gives general information about our study, solicits participation into the study, and provides links to relevant medical and educational sites. During the time period of July, 1999, to December, 2000, the site received 5, 108 visits (13 visits/day). Approximately 20 research study applications are received each month. A total of 49% (57/117) of the individuals responding to the aortic aneurysm and 63% (84/134) responding to the intracranial aneurysm study report at least two affected blood relatives in the family and, therefore, qualify for our genetic studies. In conclusion, Web-based patient recruitment is successful and provides an improved success rate due to the fact that the responders are more motivated to participate in research studies.

Sensitization following Thymoglobulin and Atgam rejection therapy as determined with a rapid enzyme-linked immunosorbent assay. US Thymoglobulin Multi-Center Study Group.

Monoclonal and polyclonal anti-thymocyte preparations play an important role in solid organ transplant immunosuppression. While it is generally accepted that blocking anti-idiotypic antibodies can decrease the efficacy of retreatment with mouse monoclonal antibody preparations, sensitization levels and subsequent effects on treatment efficacy are less clear for polyclonal preparations. Serum samples were obtained from 148 patients participating in a multicentre, double-blind randomized phase III trial comparing Atgam (Pharmacia Upjohn, horse anti-thymocyte globulin) with Thymoglobulin (SangStat Medical Corporation, rabbit anti-thymocyte globulin). Recipients of a first or second renal allograft undergoing biopsy proven acute rejection were randomized to treatment with Atgam or Thymoglobulin. Serum samples were analysed for presence of anti-thymoglobulin and anti-Atgam antibodies. Sensitization levels to rabbit IgG in Thymoglobulin-treated patients (68%, n = 54) was similar to sensitization to horse IgG in Atgam-treated patients (78%, n = 54) (two-sided p value = 0.4, Fisher's exact test), although Atgam-treated patients remained sensitized longer (at day 90, 67% anti-horse IgG positive in Atgam treated vs 24% anti-rabbit IgG in Thymoglobulin positive, p = 0.001). No difference was seen in the production of a crossreactive response. Similarly, sensitization had no significant effect on treatment success or failure. For Thymoglobulin-treated patients, the sensitization rate in successfully treated patients was 68%, while inpatients with treatment failures it was 71% (p = not significant, ns). In Atgam-treated patients, the sensitization rate in successfully treated patients was 82%, while in patients with treatment failures it was 67% (p = ns). In conclusion, patients treated with Thymoglobulin and patients treated with Atgam exhibited similar levels of sensitization, presensitization and crossreactive sensitization, although the anti-horse response was longer lasting; neither presensitization nor treatment-induced sensitization appeared to effect treatment efficacy.

Insertion and deletion mutations in the repA4 region of the IncFII plasmid NR1 cause unstable inheritance.

Mutants of IncFII plasmid NR1 that have transposons inserted in the repA4 open reading frame (ORF) are not inherited stably. The repA4 ORF is located immediately downstream from the replication origin (ori). The repA4 coding region contains inverted-repeat sequences that are homologous to the terC inverted repeats located in the replication terminus of the Escherichia coli chromosome. The site of initiation of leading-strand synthesis for replication of NR1 is also located in repA4 near its 3' end. Transposon insertions between ori and the right-hand terC repeat resulted in plasmid instability, whereas transposon insertions farther downstream did not. Derivatives that contained a 35-bp frameshift insertion in the repA4 ORF were all stable, even when the frameshift was located very near the 5' end of the coding region. This finding indicates that repA4 does not specify a protein product that is essential for plasmid stability. Examination of mutants having a nest of deletions with endpoints in or near repA4 indicated that the 3' end of the repA4 coding region and the site of leading-strand initiation could be deleted without appreciable effect on plasmid stability. Deletion of the pemI and pemK genes, located farther downstream from repA4 and reported to affect plasmid stability, also had no detectable effect. In contrast, mutants from which the right-hand terC repeat, or both right- and left-hand repeats, had been deleted were unstable. None of the insertion or deletion mutations in or near repA4 affected plasmid copy number. Alteration of the terC repeats by site-directed mutagenesis had little effect on plasmid stability. Plasmid stability was not affected by a fus mutation known to inactivate the termination function. Therefore, it appears that the overall integrity of the repA4 region is more important for stable maintenance of plasmid NR1 than are any of the individual known features found in this region.

Suppression of replication-deficient mutants of IncFII plasmid NR1 can occur by two different mechanisms that increase expression of the repA1 gene.

Replication-proficient (Rep+) revertants were isolated from mutants of IncFII plasmid NR1 that were replication defective (Rep-). The parental Rep- plasmids contained a mutation that inactivated promoter PE for transcription of RNA-E, a trans-acting repressor of translation of the essential RepA1 replication initiation protein of NR1. The PE mutation also introduced a nonsense codon into a leader peptide gene that precedes and slightly overlaps the repA1 translation initiation site in the mRNA. This reduced the rate of synthesis of RepA1 by uncoupling its translation from that of the leader peptide. The reduced rate of RepA1 synthesis was responsible for the Rep- phenotype. All Rep+ revertants retained the PE mutation and contained second-site mutations responsible for suppression of the Rep- phenotype. One Rep+ revertant contained a second mutation adjacent to the Shine-Dalgarno sequence of repA1. Another Rep+ revertant contained a mutation in the repA2 gene, which encodes the trans-acting repressor of transcription of repA1. By using translational lacZ gene fusions, it was found that both kinds of suppressor mutation increased the expression of repA1 to a level sufficient to support replication. In both cases, the synthesis of RepA1 remained uncoupled from that of the leader peptide. The Shine-Dalgarno mutation increased the rate of leader peptide-independent translation of repA1 mRNA and also reduced the sensitivity of repA1 mRNA to inhibition by RNA-E. The repA2 mutation inactivated the RepA2 repressor and increased the rate of transcription of repA1 mRNA. The translational lacZ gene fusions were used to assess the range of regulation of expression of repA1 provided by each of the RNA-E and RepA2 regulatory circuits. By constructing miniplasmids that contained various combinations of the mutations, the contributions of the RNA-E and RepA2 regulatory circuits were assessed with respect to control of plasmid copy number and stable inheritance. Plasmids that lacked either circuit were less stable than wild-type plasmids.

Expression of the repA1 gene of IncFII plasmid NR1 is translationally coupled to expression of an overlapping leader peptide.

Examination of a group of mutants of plasmid NR1 that had lost the expression of IncFII plasmid incompatibility (Inc-) revealed a group that had also lost replication proficiency (Rep-). These mutants were obtained from plasmids in which the NR1 replication control region was present in a cointegrate with plasmid pBR322. Whereas the wild-type parental cointegrate plasmid was capable of replicating in a polA host owing to the PolA independence of NR1 replication, the mutants were not able to transform a polA host. Losses of both expression of IncFII plasmid incompatibility and replication proficiency were found to result from the same single base-pair substitution in four independently isolated Inc- Rep- mutants. The mutation inactivates promoter PE for the transcription of RNA-E, a trans-acting repressor of translation of the essential RepA1 replication initiation protein of NR1. Although the loss of RNA-E synthesis had been expected to increase the expression of repA1, the efficiency of translation of repA1 mRNA from these mutants was at least 100-fold lower than that from the wild type, as revealed by repA1-lacZ translational fusions. The PE mutation introduced a stop codon into a 24-amino-acid reading frame that precedes the repA1 gene and terminates just 2 bp downstream from the repA1 start codon. This putative leader peptide was also expressed in a lacZ translational fusion, and its expression was reduced by a factor of 10(4) by the PE mutation. The expression of the leader peptide and the expression of repA1 were regulated by RNA-E. These results suggest that the expression of repA1 is coupled to the translation of the leader peptide and that the repression of repA1 translation by RNA-E may occur via inhibition of the translation of the leader peptide.

Autoregulation of the stability operon of IncFII plasmid NR1.

The stb locus of IncFII plasmid NR1, which mediates stable inheritance of the plasmid, is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins. The two tandem genes, stbA and stbB, are transcribed as an operon from promoter PAB. Using PAB-lacZ gene fusions, it was found that the stb operon is autoregulated. A low-copy-number stb+ plasmid introduced into the same cell with the PAB-lacZ fusion plasmid repressed beta-galactosidase activity about 5-fold, whereas a high-copy-number stb+ plasmid repressed beta-galactosidase about 15-fold. The details of autoregulation were analyzed by varying the concentrations of StbA and StbB to examine their effects on expression from the PAB-lacZ fusion plasmid. StbB protein by itself had autorepressor activity. Although StbA protein by itself had no detectable repressor activity, plasmids that encoded both stbA and stbB repressed more effectively than did those that encoded stbB alone. Plasmids with a mutation in stbA had reduced repressor activity. One mutation in stbB that inactivated the stability function also reduced, but did not eliminate, repressor activity. Repressor activity of the mutant StbB protein was effectively enhanced by stbA. These results indicate that StbB serves two functions, one for stable inheritance and one for autoregulation of the stb operon, both of which may be influenced by StbA protein.

Identification of a monocyte-derived factor which regulates synthesis of insulin receptors on activated T-lymphocytes (MIRRF).

The regulation of the insulin receptor on the activated T-lymphocyte was studied. It has been previously shown that the monocyte with its constitutive insulin receptor can signal the quiescent T-lymphocyte with respect to ambient insulin concentration which regulates the copies of insulin receptors synthesized during the lymphocyte activation event. In this communication it is shown that the vehicle by which the monocyte signals the T-lymphocyte is a soluble, small molecular weight protein. Initially a bioassay was established to test the putative monocyte-derived factor in which freshly prepared purified populations of monocytes were incubated with insulin, extensively washed, and replated with lymphocytes in microwells or across a 3 microns filter from lymphocytes using the appearance of insulin receptors on T lymphocytes responding to lectin as measured by a radioligand binding assay as the outcome variable. Dose response and time course relationships were established to develop the ideal conditions for the bioassay. It was shown that the monocyte-derived insulin receptor regulatory factor (MIRRF) could be readily detected in conditioned medium of insulin-incubated and then washed monocytes as a starting point for attempts at later purification. Using rats fed an essential fatty acid deficient diet (EFAD), incapable of generating standard prostanoids, it was demonstrated that the MIRRF was readily detectable in our standard bioassay revealing that the factor was not a member of the arachidonic acid family. Lastly, it was shown that MIRRF is cycloheximide sensitive and either is a protein or requires protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

The impact of acemannan on the generation and function of cytotoxic T-lymphocytes.

Acemannan, an antiviral agent with immune enhancement capabilities, was studied for its impact on cytotoxic T-lymphocyte (Tc) function generated in response to alloantigen. To investigate whether acemannan directly stimulated the generation of Tc from primary mixed lymphocyte cultures (MLC), the drug was added at the initiation of the MLC. There was a dose-related, statistical increase in killer T-cell generation produced by acemannan in the clinically relevant dose range. The lowest test dose of the drug (2.6 x 10(-9) M) increased chromium release nearly two-fold; the 2.6 x 10(-8) M dose gave a maximal 3.5 fold increase in cytotoxic T-cells. To study whether acemannan enhanced the capacity of Tc once generated to alloantigen to destroy targets bearing the sensitizing antigens, MLR were established in the absence of any drug. Acemannan at the two highest doses increased the functional capacity of Tc to destroy target cells to which they had been sensitized in the MLR. To control for the possibility that acemannan was directly cytotoxic to target cells, targets were incubated alone with drug and without sensitized killer T-cells. No dose of acemannan was found to be cytotoxic to these cells. In conclusion, acemannan did enhance the generation of cytotoxic T-cells when added at the initiation of the MLR. When acemannan was added at the completion of allostimulation, an increase of almost 50% killing by Tc was also observed. These effects can not be explained by direct drug related toxicity and suggest a functional correlate to the previously described immune enhancing properties of the agent. As this drug is being tested for the treatment of HIV infections, these data provide at least one immunologic mechanism by which acemannan may be clinically salutory.

Transcription of the stability operon of IncFII plasmid NR1.

The stability (stb) locus of IncFII plasmid NR1 is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins. The stb locus was found to be transcribed from a promoter site just upstream from the first gene, stbA. This promoter was active for transcription both in vivo and in vitro and was located within the region that includes the essential cis-acting site. Transcripts initiated from this site were approximately 1,500 to 1,600 nucleotides in length. Northern (RNA) blot analysis indicated that the transcripts traversed both stbA and the downstream gene, stbB. Mutants from which the promoter had been deleted failed to produce detectable transcripts from either stbA or stbB. Transcription of a third open reading frame, stbC, which is contained within the stbB gene in the opposite DNA strand, could not be detected. For a mutant in which a transposon had been inserted in stbA, no transcription of stbB was detected. After deletion of most of the transposon, which left behind a 35-bp frameshift insertion in stbA, transcription of stbB was restored, although the insertion still had a polar effect on stbB function. The rate of in vivo transcription of the stb locus was measured by pulse-labeling of RNA followed by quantitative RNA-DNA hybridization. Mutants deleted of stbB had an approximately 10-fold increase in the rate of transcription, whereas those deleted of the promoter region had at least a 10-fold reduction in transcription rate. The half-life of stb mRNA was approximately 2 min. These data suggest that stbA and stbB are cotranscribed as an operon that may be autoregulated.

Immunobiological consequence of regulation of insulin receptor on alloactivated lymphocytes in normal and obese subjects.

Acute manipulations of insulin in vivo regulate the display of insulin receptors induced on activated T lymphocytes after presentation of alloantigen. This study explored the immunobiological consequences of regulation of insulin-receptor display by acute manipulations of insulin achieved during the hyperinsulinemic-euglycemic clamp in healthy normal individuals and obese subjects. T lymphocytes were isolated at 0, 1, and 4 h of hyperinsulinemia from seven normal volunteers and seven obese individuals and studied for their capacity to 1) synthesize a complement of insulin receptors on cell membrane, 2) respond to alloantigen in the mixed-lymphocyte culture (an immunologic activity unrelated to manipulations in insulin concentrations in complete medium), and 3) respond to the lymphocyte-mediated cytotoxicity reaction (an immunologic activity known to be modulated by insulin). In the face of a reduction in receptor numbers to 25% of baseline in normal individuals, alloreactivity in the mixed-lymphocyte culture was not affected (95 +/- 9% of time 0 after 4 h of hyperinsulinemia), whereas lymphocyte-mediated cytotoxicity fell from 14 +/- 4 at time 0 to 2 +/- 2% sp Cr release (P less than 0.02). Hyperinsulinemia achieved by the clamp in seven obese subjects did not alter the synthesis of insulin receptors on cell membrane after presentation of alloantigen. In the absence of further reduction of insulin-receptor membrane display, neither the mixed-lymphocyte culture nor lymphocyte-mediated cytotoxicity reaction was affected. It is concluded that those immunologic activities of lymphocytes that can be modulated by insulin are affected by regulation of insulin-receptor display on activated lymphocytes. Therefore, receptor regulation is not effete but carries significant immunologic consequence.(ABSTRACT TRUNCATED AT 250 WORDS)

DnaA protein is not essential for replication of IncFII plasmid NR1.

By transformation of dnaA null mutant host cells that are suppressed either by an rnh mutation or by chromosomal integration of a mini-R1 plasmid, it was shown that replication of miniplasmids composed of the NR1 minimal replicon had no absolute dependence upon DnaA protein. In addition, the suppression of the dnaA null mutation by the integrated mini-R1, which is an IncFII relative of NR1, was found to be sensitive to the expression of IncFII-specific plasmid incompatibility. This suggests that the integrative suppression by mini-R1 is under the control of the normal IncFII plasmid replication circuitry. Although NR1 replication had no absolute requirement for DnaA, the copy numbers of NR1-derived miniplasmids were lower in dnaA null mutants, and the plasmids exhibited a much reduced stability of inheritance during subculture in the absence of selection. This suggests that DnaA protein may participate in IncFII plasmid replication in some auxiliary way, such as by increasing the efficiency of formation of an open initiation complex at the plasmid replication origin. Such an auxiliary role for DnaA in IncFII replication would be different from that for replication of most other plasmids examined, for which DnaA has been found to be either essential or unimportant.

DNA bending near the replication origin of IncFII plasmid NR1.

The DNA replication origin of plasmid NR1 is located approximately 190 base pairs downstream from the 3' end of the repA1 gene, which encodes the essential initiation protein for replication of the plasmid. Restriction endonuclease fragments that contain the NR1 replication origin and its flanking sequences at circularly permuted positions were obtained by digesting oligomers of ori-containing DNA fragments with sets of enzymes that each cut only once in every ori fragment. Polyacrylamide gel electrophoresis of these permuted restriction fragments showed anomalous mobilities, indicating the presence of a DNA bending locus. Through analysis of the relative mobility plots of these permuted fragments, we found one or two possible DNA bending sites located in the intervening region between the repA1 gene and the replication origin of NR1. It seems possible that DNA bending in this region might help to orient the replication origin alongside the repA1 gene, which could contribute to the cis-acting character of the RepA1 initiation protein.

Complementation of mutants of the stability locus of IncFII plasmid NR1. Essential functions of the trans-acting stbA and stbB gene products.

A series of unstable mutants of the stability (stb) locus of IncFII plasmid NR1 was subjected to a complementation analysis. The mutant collection included plasmids with point, insertion and deletion mutations in stb. These mutations affected the tandem genes stbA and stbB, which encode stability proteins StbA and StbB, or the PAB transcription promoter, which is located upstream from stbA in a region that contains an essential cis-acting site. Deletion mutants that lacked the region containing promoter PAB could not be complemented (stabilized) by providing StbA and StbB in trans. Deletion mutants that lacked stbA and stbB but retained the PAB region were complemented in trans but required both StbA and StbB, indicating that both proteins were essential for stable inheritance. stbA- point mutants were complemented in trans by either wild-type or stbA+ stbB- clones of the stability region. However, mutants with insertions in stbA were complemented only by wild-type clones, which suggested the insertions were polar on expression of the downstream stbB gene. A plasmid with a stbB- point mutation was complemented in trans by wild-type but not by stbA- stbB+ clones. In addition, plasmid clones that expressed StbB in the absence of StbA caused destabilization of (were incompatible with) stb+ derivatives of NR1 in trans, whereas clones that expressed only wild-type StbA or both StbA plus StbB did not. Plasmid clones that contained only the essential cis-acting PAB region did not cause destabilization of stb+ plasmids in trans. These results suggest that an excess of StbB protein provided in trans may cause a depletion of the essential StbA protein. Therefore, these results may be consistent with the hypothesis that StbB is an autorepressor of the stbAB operon.