A new approach to the cloning of genes encoding T-cell epitopes

The molecular structure of antigens recognized exclusively by T cells, such as minor histocompatibility antigens and some antigens that provoke autoimmune responses, has proved difficult to determine. Recently, several antigens induced on tumor cells by mutagen treatment have been cloned by transfection of genomic DNA libraries into P1.HTR cells, screening for antigen expression using T-cell clones, and subsequent recovery of the integrated DNA by cosmid rescue. We have modified this techniques and have stably transfected P1. HTR cell lines with polyoma T antigen, which allows episomal replication of the shuttle vector, pCDM8. Using pCDM8-CAT constructs, we have determined the frequency of transfection and plasmid copies taken up per cell under optimal transfection conditions. Using a pCDM8 construct which expresses the tumor-specific antigen, P91A (pCDM8-tum^-), that is recognized by a T-cell clone, we have found that cells transfected with this antigen can be recognized by the T-cell clone when they are present at only 1%–3% of a mixed population. Progeny of a single cell transfected with pCDM8-tum^-: pCDM8-CAT at proportions of 1:10, 1:25, and 1:50 are recognized by the T-cell clone. Furthermore, Hirt extracted plasmid DNA from transfectants expressing the tum^- antigen can be amplified in bacteria, transfected back into P1.HTR recipients, and recognized by the T-cell clone. This approach should enable reasonably rapid screening of cDNA libraries for even relatively low abundance messages encoding, for example, minor histocompatibility and allonatigens, and allow their subsequent cloning. Address correspondence and offprint requests to: D. M. Scott.     

A practical approach to crosslinking

The various aspects of chemical crosslinking are addressed. Crosslinker reactivity, specificity, spacer arm length and solubility characteristics are detailed. Considerations for choosing one of these crosslinkers for a particular application are given as well as reaction conditions and practical tips for use of each category of crosslinkers.Abbreviations ABH azidobenzoyl hydrazide - ANB- NOS N-5-azido-2-nitrobenzoyloxysuccinimide - ASIB 1-(p-azidosalicylamido)-4-(iodoacetamido)butane - ASBA 4-(p-azidosalicylamido)butylamine - APDP N-[4-(p-azidosalicylamido) butyl]-3(2-pyridyldithio)propionamide - APG p-azidophenyl glyoxal monohydrate - BASED bis-[-(4-azidosalicylamido)ethyl] disulfide - BMH bismaleimidohexane - BS³ bis(sulfosuccinimidyl) suberate - BSOCOES bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone - DCC N,N-dicyclohexylcarbodiimide - DFDNB 1,5-difluoro-2,4-dinitrobenzene - DMA dimethyl adipimidate·2HCl - DMP dimethyl pimelimidate·2HCl - DMS dimethyl suberimidate·2HCl - DPDPB 1,4-di-(3,2-pyridyldithio)propionamido butane - DMF dimethylformamide - DMSO dimethylsulfoxide - DSG disuccinimidyl glutarate - DSP dithiobis(succinimidylpropionate) - DSS disuccinimidyl suberate - DST disuccinimidyl tartarate - DTSSP 3,3-dithiobis (sulfosuccinimidylpropionate) - DTBP dimethyl 3,3-dithiobispropionimidate·2HCl - EDC or EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride - EDTA ethylenediaminetetraacetic acid disodium salt, dihydrate - EGS ethylene glycolbis(succinimidylsuccinate) - GMBS N--maleimidobutyryloxysuccinimide ester - HSAB N-hydroxysuccinimidyl-4-azidobenzoate - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester - MES 4-morpholineethanesulfonic acid - NHS N-hydroxysuccinimide - NHS-ASA N-hydroxysuccinimidyl-4-azidosalicylic acid - PMFS phenylmethylsulfonyl fluoride - PNP-DTP p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate - SAED sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide) ethyl-1,3-dithiopropionate - SADP N-succinimdyl (4-azidophenyl)1,3-dithiopropionate - SAND sulfosuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl-1,3-dithiopropionate - SANPAH N-succinimidyl-6(4-azido-2-nitrophenyl-amino)hexanoate - SASD sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3-dithiopropionate - SATA N-succinimidyl-S-acetylthioacetate - SDBP N-hydroxysuccinimidyl-2,3-dibromopropionate - SIAB N-succinimidyl(4-iodoacetyl)aminobenzoate - SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate - SMPB succinimidyl 4-(p-maleimidophenyl) butyrate - SMPT 4-succinimidyloxycarbonyl--methyl--(2-pyridyldithio)-toluene - sulfo-BSOCOES bis[2-sulfosuccinimidooxycarbonyloxy) ethyl]sulfone - sulfo-DST disulfosuccinimidyl tartarate - sulfo-EGS ethylene glycolbis(sulfosuccinimidylsuccinate) - sulfo-GMBS N--maleimidobutyryloxysulfosuccinimide ester - sulfo-MBS m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester - sulfo-SADP sulfosuccinimidyl(4-azidophenyldithio)propionate - sulfo-SAMCA sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate - sulfo-SANPAH sulfosuccinimidyl 6-(4-azido-2-nitrophenylamino)hexanoate - sulfo-SIAB sulfosuccinimidyl(4-iodoacetyl)aminobenzoate - sulfo-SMPB sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate - sulfo-SMCC sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate - SPDP N-succinimidyl 3-(2-pyridyldithio)propionate     

A novel and rapid cloning method for the T-cell receptor variable region sequences

Conventional polymerase chain reactions (PCR) require sequence information on both ends of the DNA to be amplified. The novel technique described here allows the amplification of cDNA fragments with sequence information from one end only. Blunt-ended double-strand cDNA is prepared, circularized with T4 DNA ligase and used as a PCR template. The two PCR primers are desinged to hybridize to the known region in an outward orientation allowing the amplification of the unknown sequence. The method was established using the -chain of T-cell antigen receptors (Tcr) as an example. The cDNA synthesized from 1 g of total RNA from human peripheral lymphocytes was amplified and cloned resulting in a library of 1–2 × 10⁶ Tcr-specific clones. The method should also be useful for cloning full-length cDNA or for the identification of new members of a gene family that share a conserved domain.     

A practical approach to quantitate hepatic excretory function

A statistical comparison of different BSP tests was carried out in normal subjects and in patients with various degrees of chronic liver damage. Only the logarithm of the BSP retention correlated linearly with the physiologically more meaningful determination of the maximal excretory capacity of the liver (BSP Tm, Wheele''s method). A double logarithmic transformation was required to correlate the second exponential component of the BSP plasma disappearance curve (k₂) with the BSP Tm. When the limitations of these methods are kept in mind, the observed statistical relationships can be used to express hepatic functional deterioration in more physiological terms.     

A practical false discovery rate approach to identifying patterns of differential expression in microarray data

SUMMARY: Searching for differentially expressed genes is one of the most common applications for microarrays, yet statistically there are difficult hurdles to achieving adequate rigor and practicality. False discovery rate (FDR) approaches have become relatively standard; however, how to define and control the FDR has been hotly debated. Permutation estimation approaches such as SAM and PaGE can be effective; however, they leave much room for improvement. We pursue the permutation estimation method and describe a convenient definition for the FDR that can be estimated in a straightforward manner. We then discuss issues regarding the choice of statistic and data transformation. It is impossible to optimize the power of any statistic for thousands of genes simultaneously, and we look at the practical consequences of this. For example, the log transform can both help and hurt at the same time, depending on the gene. We examine issues surrounding the SAM 'fudge factor' parameter, and how to handle these issues by optimizing with respect to power.     

Hamster estrogen receptor cDNA: cloning and mRNA expression

Estrogen-induced hamster kidney tumor model serves as a useful model to study the biochemical and molecular mechanisms of hormonal carcinogenesis. In this model, we have demonstrated an increased expression of estrogen receptor mRNA and protein in estrogen-treated kidneys and in estrogen-induced tumors. The sequence information for hamster estrogen receptor gene is not known and has been investigated in this study. A hamster uterus cDNA library was constructed and the 5'-region of the hamster estrogen receptor cDNA cloned from this library using polymerase chain reaction (PCR) methodology. Additionally, hamster kidney polyadenylated RNA was reverse transcribed and PCR amplified using primers that were designed based on maximum homology between mouse, rat and human estrogen receptor cDNAs. These PCR amplified fragments were cloned into plasmid vectors and clones with the expected size of the insert subjected to Southern blot analysis using human estrogen receptor cDNA as a probe. The positive clones on Southern blot analysis and the PCR amplified products from these clones were subjected to DNA sequence analysis. Using this strategy, a full length, 1978 bp hamster estrogen receptor cDNA has been cloned which shows 87% homology with human, 90% with rat and 91% with mouse estrogen receptor cDNA. The deduced amino acid shares 88% homology with human, and 93% with rat and mouse estrogen receptors. Hamster estrogen receptor domain C (DNA binding domain) shows a 100% homology with a similar domain from mouse, rat, human, pig, sheep, horse and chicken estrogen receptor (Genebank reference ID: AF 181077).     

Molecular cloning and functional expression of a VIP-specific receptor

Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: PAC(1), with high affinity for PACAP, and VPAC(1) and VPAC(2) with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor (VIP(s)) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial NH(2)-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a VPAC(2) receptor from GP gastric smooth muscle and VIP(s) from GP teniae coli smooth muscle. The cDNA sequence of the VIP(s) encodes a 437-amino acid protein (M(r) 49,560) that possesses 87% similarity to VPAC(2) receptors in rat and mouse and differs from the VPAC(2) receptor in GP gastric smooth muscle by only two amino-acid residues, F(40)F(41) in lieu of L(40)L(41). In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity (IC(50) 1.4 nM) and stimulated cAMP formation with high potency (EC(50) 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity (IC(50) 2.3 nM) and stimulated cAMP with equally high potency (EC(50) 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a VIP(s) distinct from VPAC(1) and VPAC(2) receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues (L(40)L(41)) in the NH(2)-terminal ligand-binding domain.     

Molecular cloning and expression of the human interferon-gamma receptor

A cDNA encoding the human interferon-gamma receptor was isolated from a lambda gt11 expression library using a polyclonal antireceptor antiserum. The gene for this receptor was identified in a cosmid library and transfected into mouse cells. The human interferon-gamma receptor expressed in mouse cells displayed the same binding properties as in human cells. However, transfected cells were not sensitive to human IFN-gamma, suggesting the need for species-specific cofactors in receptor function. As inferred from the cDNA sequence, the human interferon-gamma receptor shows no similarities to known proteins and represents a novel transmembrane receptor. It is most likely the product of a single mRNA and a gene located on chromosome 6q.     

An approach to practical persistence

 A basic problem in community ecology is determining whether a community of interacting species will survive in the long term. A criterion ensuring this is that of permanence (or uniform persistence), which is based on the idea that species densities for large time are above minimum non-zero levels. There are various mathematical techniques for investigating permanence, but they do not yield an estimate for the minimum levels, and these may lie below minimum viability levels in the biological sense of ‘Practical Persistence’. Here we study a technique for obtaining explicit expressions for the minimum levels when one species is ‘slow’. This is illustrated for a predator–prey problem governed by difference equations, and we note that the technique is applicable even when the dynamics is chaotic.. Received: 7 April 1997 / Revised version: 25 March 1998     

A genome annotation-driven approach to cloning the human ORFeome

We have developed a systematic approach to generating cDNA clones containing full-length open reading frames (ORFs), exploiting knowledge of gene structure from genomic sequence. Each ORF was amplified by PCR from a pool of primary cDNAs, cloned and confirmed by sequencing. We obtained clones representing 70% of genes on human chromosome 22, whereas searching available cDNA clone collections found at best 48% from a single collection and 60% for all collections combined.     

Production activity control: A practical approach to scheduling

There is a widely perceived gap within the domain of scheduling for manufacturing systems, namely, many of the methods employed by production supervisors are quite different from those developed by researchers. In a sense, this inconsistency highlights the important fact that much scheduling research has failed to win approval where it matters most, namely, within the manufacturing system. In this article, we argue for a practical approach to scheduling for manufacturing systems, one that we believe can narrow, and possibly bridge, the gap between theory and practice. This approach is based upon a well-defined and modular architecture for scheduling, termedproduction activity control. This architecture is the foundation of our proposed solution to scheduling, since it provides a coherent blueprint for the synthesis of information technology and scheduling strategies. The result of this synthesis is a design tool for production activity control, which allows for detailed and disciplined experimentation with a range of scheduling strategies in a controlled and simulated environment. Due to the unique modular property of the design tool, these strategies may then be implemented live in a flexible manufacturing facility, hence narrowing the gap between scheduling theory and manufacturing practice. Our overall approach is tested through an appropriate implementation in a modern electronics assembly plant.     

Molecular cloning and functional expression of a Drosophila corazonin receptor

Here we report a novel method for selecting human antibody fragments from nonimmunized variable domain libraries. The antibody fragments are selected on the basis of stabilization of the variable domain fragment (F(v)) in the presence of target antigens ("open sandwich selection"). One variable domain is displayed on phages and another is prepared as soluble molecules. These two reagents are mixed with the biotinylated target molecule and ternary complexes are captured by using streptavidin-conjugated magnet beads. After extensive washing, enriched clones are eluted by using target antigen. Some of the clones selected after 3 rounds are prepared as soluble domains, which then undergo another selection process. We obtained several human antibody fragments specific for human soluble erythropoietin receptor by using this method. Our method minimizes several of the disadvantages associated with human antibody selection through a phage-display system, such as construction of a large-scale library, deletion of genes during selection, and nonspecific binding.     

Molecular cloning and expression of the murine interleukin-5 receptor

Murine interleukin-5 (IL-5) is known to play an essential role in Ig production of B cells and proliferation and differentiation of eosinophils. Here, we have isolated cDNA clones encoding a murine IL-5 receptor by expression screening of a library prepared from a murine IL-5 dependent early B cell line. A cDNA library was expressed in COS7 cells and screened by panning with the use of anti-IL-5 receptor monoclonal antibodies. The deduced amino acid sequence analysis demonstrates that the receptor is a glycoprotein of 415 amino acids (Mr 45,284), including an N-terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tail (54 amino acids). COS7 cells transfected with the cDNA expressed a 60 kd protein that bound IL-5 with a single class of affinity (KD = 2-10 nM). FDC-P1 cells transfected with the cDNA for murine IL-5 receptor showed the expression of IL-5 binding sites with both low (KD = 6 nM) and high affinity (KD = 30 pM) and acquired responsiveness to IL-5 for proliferation, although parental FDC-P1 cells did not show any detectable IL-5 binding. In addition, several cDNA clones encoding soluble forms of the IL-5 receptor were isolated. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected in cell lines that display binding sites for murine IL-5. Homology search for the amino acid sequence of the IL-5 receptor reveals that the IL-5 receptor contains a common motif of a cytokine receptor family that is recently identified.     

Purification, cloning, and expression of the prolactin receptor

The rat liver prolactin receptor has been purified to homogeneity, and partial amino acid sequences have been obtained. The structure of the receptor has been deduced from a single complementary DNA clone. The mature protein of 291 amino acids has a relatively long extracellular region, a single transmembrane segment, and a short (57 amino acids) cytoplasmic domain. With the rat cDNA used as a probe, the prolactin receptor in rabbit mammary gland and human hepatoma cells has also been isolated. These tissues contain a second, longer form of the receptor (592 and 598 amino acids, respectively). Both the short and long forms of the prolactin receptor show regions of strong sequence identity with the human and rabbit growth hormone receptors, suggesting that the prolactin and growth hormone receptors originate from a common ancestor.     

Molecular cloning and expression of a fifth muscarinic acetylcholine receptor

A cDNA of 2149 base pairs with an incomplete open reading frame (ORF) encoding amino acids 1-516 of a 531-amino acid protein highly homologous to muscarinic receptors was cloned from a rat brain cDNA library. The complete ORF was then deduced from a DNA fragment cloned from a rat genomic library. This ORF was subcloned into the eukaryotic expression vector p91023(B) under control of the adenovirus major late promoter and co-transfected with the thymidine kinase selection marker into muscarinic receptor-negative, thymidine kinase-negative murine L cells. Stable transformants were selected and tested for acquisition of muscarinic receptors by following appearance of specific binding sites for the muscarinic ligand [3H] N-methylscopolamine. Two cell lines, LM5.36 and LM5.40, were cloned and shown to express typical muscarinic receptor sites, thus confirming that the newly cloned ORF encodes a muscarinic receptor, the rat M5 muscarinic acetylcholine receptor. Tests for activities showed it to stimulate phosphoinositide hydrolysis in intact cells, without affecting positively or negatively adenylyl cyclase activity. The M5 receptor contains two putative glycosylation sites at its amino terminus and, based on hydropathicity analysis, is predicted to span the plasma membrane seven times. Like 17 other receptors of this class, the M5 receptor has 19 conserved amino acids, among which are 4 prolines located in the 4th, 5th, 6th, and 7th predicted transmembrane regions, conferring possible bends to these helices, and 2 cysteines, one in the 1st and the other in the 2nd extracellular loop, possibly providing for a disulfide bond. Similarity in amino acid composition and in patterns of antagonist binding and biologic effects suggest the M5 receptor to be M1-like.