Designating eukaryotic orthology via processed transcription units.

Orthology is a widely used concept in comparative and evolutionary genomics. In addition to prokaryotic orthology, delineating eukaryotic orthology has provided insight into the evolution of higher organisms. Indeed, many eukaryotic ortholog databases have been established for this purpose.

Down-regulation of human NDR gene in megakaryocytic differentiation of erythroleukemia K562 cells.

To study the control of hematopoietic cell differentiation, a human negative differentiation regulator (NDR) gene was identified by the comparative analysis of differentially expressed genes in hemato-lymphoid tissues. NDR is expressed preferentially in the adult bone marrow, fetal liver and testis. Immunocytochemistry with anti-NDR antiserum showed the presence of NDR in human erythroleukemia K562 cell line and CD34+ cells sorted from the umbilical cord blood.

A complexity reduction algorithm for analysis and annotation of large genomic sequences.

DNA is a universal language encrypted with biological instruction for life. In higher organisms, the genetic information is preserved predominantly in an organized exon/intron structure. When a gene is expressed, the exons are spliced together to form the transcript for protein synthesis.

Identification of novel human genes evolutionarily conserved in Caenorhabditis elegans by comparative proteomics.

Modern biomedical research greatly benefits from large-scale genome-sequencing projects ranging from studies of viruses, bacteria, and yeast to multicellular organisms, like Caenorhabditis elegans. Comparative genomic studies offer a vast array of prospects for identification and functional annotation of human ortholog genes. We presented a novel comparative proteomic approach for assembling human gene contigs and assisting gene discovery.

S- and G2-phase cell cycle arrests and apoptosis induced by ganciclovir in murine melanoma cells transduced with herpes simplex virus thymidine kinase.

Mechanism of cell killing by transfer of Herpes simplex virus type-1 thymidine kinase (HSVtk) and subsequent ganciclovir (GCV) treatment was examined in B16F10 murine melanoma model. While parental B16F10 melanoma cells were resistant to GCV at 100 microM or higher, HSVtk-transduced B16F10 melanoma cell clones became susceptible to GCV with IC50 of 0.1 to 0.

Identification and characterization of a human Vlambda5 (T1) germline gene that encodes structurally unique lambda light chains.

The human germline Vlambda repertoire consists of about 30 functional genes that have been classified into 10 families on the basis of homologies in nucleotide sequences that encode approximately the first 96 to 104 residues of lambda light chains. One family, termed Vlambda5, is of special interest because the lambda light chain products of these genes have unique structural features. We have now isolated from genomic DNA one member of this family, designated IGLV5-1, using as a molecular probe a partial Vlambda5-germline-gene fragment generated by polymerase chain reaction.

Distinctive serologic, chemical, and molecular properties of human lambda IV light chains.

Through extensive serologic, chemical, and molecular studies involving monoclonal Ig proteins and B cell-related populations, we provide definitive evidence that the V lambda IV subgroup of human light (L) chains is separate and distinct from V lambda III and all other known V lambda gene families. lambda IV and lambda III L chains were differentiated immunochemically using well-characterized polyclonal and monoclonal anti-V lambda subgroup-specific Abs. Prototypic L chains, originally classified as lambda IV on the basis of distinctive framework region 1 residues, were distinguished serologically from lambda IIIa, lambda IIIb, and lambda IIIc proteins and also from lambda I, lambda II, lambda VI, and lambda VIII L chains.

Molecular characterization of a human V lambda VIII germline gene.

Human lambda light chains of the recently recognized variable region (VL) subgroup V lambda VIII can be distinguished from proteins of other V lambda gene families on the basis of distinctive chemical and serologic properties and by their preferential association with certain types of autoantibodies, i.e. rheumatoid factors (RFs).

Detection of delta F508 mutation of the cystic fibrosis gene by matrix-assisted laser desorption/ionization mass spectrometry.

The most common mutation of the cystic fibrosis gene is characterized by the deletion of three nucleotides that code phenylalanine in the 508 position of the cystic fibrosis transmembrane conductance regulator. We report the first measurements by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry for the delta F508 mutation in cystic fibrosis carriers and patients. Furthermore, in a blind test, results from the normal and delta F508 mutant alleles in 30 clinical samples based on MALDI mass spectrometry and on conventional gel analysis of the DNA were in total agreement.

3-Aminopicolinic acid as a matrix for laser desorption mass spectrometry of biopolymers.

3-Aminopicolinic acid (3-APA) was tested and found to be a useful matrix for matrix-assisted laser desorption/ionization of DNA and protein. Single-stranded DNA segments of 150-mer and double-stranded DNA of 246 base pairs were successfully detected by using 3-APA as an ultraviolet-absorbing matrix in a linear time-of-flight mass spectrometer. In the case of the double-stranded DNA, only parent ions corresponding to single-stranded DNA were observed.

Identification and characterization of a functional human Ig V lambda VI germline gene.

We have isolated from a human genomic library a potentially functional and distinctive germline gene, designated IGLV6S1, that encodes for light chains of the V lambda VI subgroup. An identical germline gene was cloned from fibroblasts obtained from a patient with light-chain-associated amyloidosis (AL amyloidosis) whose serum and urine contained, respectively, a monoclonal IgG lambda VI protein and a lambda VI Bence Jones protein. Isolation and characterization of cDNA cloned from the patient's bone marrow-derived monoclonal plasma cells revealed that the nucleotide and predicted protein sequences of the rearranged gene were approximately 95% and approximately 90% homologous to those of the germline gene, respectively.

Effect of maternal aging on transgene heritability in transgenic founder mice derived from zygotes microinjected with retroviral long terminal repeat-containing recombinant deoxyribonucleic acid.

To determine the stability of artificially introduced recombinant DNA in the mouse germline throughout the reproductive life, founder mice derived from fertilized eggs injected with retroviral long-terminal-repeat-containing recombinant DNAs were mated with congenic FVB/N mice. Tail DNA of all progeny were screened and restriction fragment patterns of the transgenes were examined. Litter size and percentage of transgene transmission at various reproductive age periods were analyzed.

Carbon tetrachloride induction of rapid changes in liver nuclear protein factors capable of sequence-specific binding to regulatory elements in the long terminal repeat of polytropic-class endogenous murine leukemia virus-related proviruses.

Treatment of mice with hepatic carcinogens, including CCl4, has been shown to rapidly enhance the transcription of endogenous murine leukemia virus-related proviral sequences in the liver. To understand the mechanism for this transcriptional stimulation, we used nuclear protein preparations from mouse livers to perform DNase I protection analyses and identified nuclear protein binding on approximately 20 individual sequences within the regulatory regions of the long terminal repeat (LTR) of a polytropic-class endogenous provirus clone. From 3 to 144 h after treatment with CCl4, the livers of FVB/N mice were analyzed for specific nuclear protein binding to the LTR DNA.

Insulin increases transcription of rat gene 33 through cis-acting elements in 5'-flanking DNA.

Gene 33 is a multihormonally-regulated rat gene whose transcription is rapidly and markedly enhanced by insulin in liver and cultured hepatoma cells. To examine the mechanism by which insulin regulates transcription, we have constructed chimeric plasmids in which expression of the bacterial cat gene, encoding chloramphenicol acetyltransferase (CAT), is governed by gene 33 promoter elements and contiguous sequences in DNA flanking the transcription start point (tsp). When transfected into H4IIE hepatoma cells, these constructs gave rise to stably transformed cell lines producing the bacterial CAT enzyme.

Possible involvement of the long terminal repeat of transposable element 17.6 in regulating expression of an insecticide resistance-associated P450 gene in Drosophila.

P450-A and P450-B are electrophoretically defined subsets of cytochrome P450 enzymes in Drosophila melanogaster. P450-A is present among all strains tested, whereas expression of P450-B is associated with resistance to insecticides. Monoclonal antibodies were used to obtain cDNA clones for an enzyme from each P450 subset (i.

The beta 4 subunit of the integrin family is displayed on a restricted subset of endothelium in mice.

More than 15 subunits of the integrin family of cell surface adhesion molecules have been identified. The alpha 6 beta 4 integrin has recently been identified as a component of hemidesmosomes of stratified squamous epithelium. The monoclonal antibody (mAb) 346-11A binds to the beta 4 subunit in mice.

Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.

Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active.

Molecular cloning and analysis of full-length cDNAs cognate to a rat gene under multihormonal control.

Gene 33 is a multihormonally regulated rat gene whose transcription is rapidly and markedly enhanced by glucocorticoids, insulin, or cAMP. A cDNA clone (p216) containing a nearly full-length DNA complementary to the mRNA of this gene was isolated from a cDNA library and sequenced. The cDNA represents the entire mRNA transcript, except for three bases at the 5' terminus.

Specific sequence deletions in two classes of murine leukemia virus-related proviruses in the mouse genome.

Characteristic long terminal repeats (LTR) of approximately 700 and 750 bp were found, respectively, in the two classes (polytropic and modified polytropic) of murine leukemia virus (MuLV)-related nonecotropic nonxenotropic proviral sequences in eight individual molecular clones of RFM/Un mouse chromosomal DNA fragments. Three proviral clones, two polytropic and one modified polytropic, contained sequence deletions in the viral structural genes. Nucleotide sequence analysis revealed that 7-bp direct repeats occur at both ends of deleted sequences in intact structures and one of the repeats remains in genomes with the deletion.

Characterization of two solitary long terminal repeats of murine leukemia virus type that are conserved in the chromosome of laboratory inbred mouse strains.

Twenty molecular clones containing sequences homologous to the long terminal repeats (LTRs) of the endogenous ecotropic murine leukemia virus (MuLV) of the RFM/Un mouse were isolated from a library of RFM/Un mouse spleen DNA in phage lambda. Three of these LTRs were not associated with any viral structural genes. Nucleotide sequence analysis demonstrated that they were solitary LTRs which were flanked by 4-bp directly repeated cellular sequences and which lacked primer binding sites.

Cutaneous sensitivity induced by immunization with irradiated Schistosoma mansoni cercariae. II. Autoimmunoregulation of cell-mediated cutaneous sensitivity and its reversal.

C57BL/6 mice sensitized by abdominal, dermal exposure to irradiated (50 kR) Schistosoma mansoni cercariae develop partial protection against subsequent exposure with unattenuated cercariae and express cell-mediated cutaneous sensitivity upon challenge with irradiated cercariae. Autoimmunoregulation occurs as a part of this sensitization, and can be demonstrated by augmentation of cutaneous sensitivity upon use of appropriate regimens of cyclophosphamide. Mice exposed to irradiated cercariae by either intraperitoneal or ear pinna routes developed a transient hyporesponsiveness to cercarial challenge.

Cutaneous sensitivity induced by immunization with irradiated Schistosoma mansoni cercariae. I. Induction, elicitation, and adoptive transfer analysis of cell-mediated cutaneous sensitivity.

Exposure of C57BL/6 mice to highly irradiated (50 kR) cercariae of Schistosoma mansoni leads to the development of partial resistance against subsequent challenge with unattenuated cercariae. We have analyzed the cellular immune responses that occur during the afferent and efferent phases of this protective sensitization. Mice were immunized by exposure to irradiated S.