Anchored Polymerase Chain Reaction Research Articles

Sensitive Detection of Clonal Antigen Receptor Gene Rearrangements in Non-Hodgkin’s Malignant Lymphoma With an Anchored Polymerase Chain Reaction–Based Strategy

The identification of minimal residual disease (MRD) in non-Hodgkin's malignant lymphoma is of immediate clinical relevance. Although several polymerase chain reaction (PCR)-based strategies are currently available, they all share serious technical or theoretic limitations with respect to their ability to detect the broad spectrum of mature B-cell neoplasia. We report a new strategy based on anchored PCR that does not rely on a particular chromosomal translocation or VH consensus sequence. It can yield the complete DNA sequence of the immunoglobulin heavy chain variable region (VHDJH). The assay is capable of detecting one neoplastic cell among 10(3)-10(4) normal cells with high specificity and has the potential to greatly expand the variety of B-cell malignant neoplasms that can be studied.

Characterization of the promoter for the human P-selectin gene.

P-selectin, an adhesion receptor for leukocytes, is synthesized selectively by megakaryocytes and endothelial cells. We have cloned the 5'-flanking region of the human P-selectin gene and conducted a preliminary analysis of its features. As determined by primer extension, RNase protection, and anchored polymerase chain reaction cloning, there were multiple transcriptional initiation sites from -95 to -25 nucleotides relative to the start of protein-coding sequence. Transfection of bovine aortic endothelial cells with serially truncated segments of the 5'-flanking region linked to luciferase indicated that the sequence from -249 to -13 was sufficient to promote high level gene expression. Deletions to -197, -147, and -128 gradually reduced expression to basal levels, and further deletion to -100 abolished expression. The sequence from -309 to -13 supported only basal luciferase expression in COS-7, 293, or HeLa cells. Putative regulatory elements in the short 5'-flanking sequence included a CACCC sequence, two inverted repeats similar to binding sites for the ETS and NF-kappa B/rel families, a GATA motif, and a sequence related to the GT-IIC element of the SV40 enhancer. The GATA element was functional, as it bound recombinant GATA-2, and mutations in the core sequence impaired both nuclear protein binding and gene expression. These data suggest that the P-selectin gene is regulated by a combination of cis elements and their cognate transcription factors.

In vivo clonal dominance and limited T-cell receptor usage in human CD4+ T-cell recognition of house dust mite allergens.

Sensitivity to house dust mite antigens in atopic individuals is a major cause of allergic diseases, ranging from asthma to rhinitis and dermatitis. We have studied the T-cell receptor (TCR) usage of house-dust-mite-specific CD4+ T-cell clones isolated from an atopic individual, by using the anchored polymerase chain reaction, and have analyzed the peripheral TCR repertoire of the same individual. Several T-cell clones had identified TCRs at the sequence level, despite the fact that they had been independently isolated, in some cases, in different years. These data suggest the presence in vivo of long-lived T-cell clones. We have also shown that junctional sequences identical to these clones are present in peripheral blood T cells taken 6 years after the isolation of the T-cell clones. The analysis of TCR genes used by the panel of clones reveals oligoclonality, with the variable (V) region gene segments V alpha 8 and V beta 3 being dominant, although there is minimal conservation of junctional sequences. The results have implications for understanding the TCR recognition of an environmental aeroallergen and the life span of T-cell clones in vivo during a chronic immune response.

The use of anchored polymerase chain reaction for the study of large numbers of human T-cell receptor transcripts

Anchored-PCR (A-PCR) is an approach designed to amplify and clone sequences with unknown 5' or 3' extremities. A-PCR is therefore appropriate for studying variable region of T-cell receptors (TCRs) expressed in polyclonal T-cell populations since it does not prejudge which variable gene segments are actually being used. We report here some critical modifications in the initial procedure to make it easy to clone and sequence large series of TCR transcripts. They have been introduced to improve both the yield and specificity of TCR amplified products and include re-amplification, size selections of the material combined with the successive use of nested TCR constant region specific primers. This procedure has been successfully applied to the study of the repertoire of both TCR γ/β +andγ/δ + human T-cells. The efficiency of the present A-PCR protocol will help to precisely analyze TCR usage in normal and pathological situations.

The expressed T cell receptor V gene repertoire of rheumatoid arthritis monozygotic twins: rapid analysis by anchored polymerase chain reaction and enzyme-linked immunosorbent assay.

Because of heterogeneity in the outbred human population, it has been difficult to determine the genetic factors that influence the expressed T cell receptor (TcR) repertoire in autoimmune diseases. To overcome this problem, we have developed a combination of anchored polymerase chain reaction (APCR) and enzyme-linked immunosorbent assay (ELISA) that can accurately assess TcR V gene frequencies in numerous clinical samples. The results are independent of amplification efficiency, and V gene usage can be readily analyzed with an ELISA plate reader and associated software. Using this method, the TcR V beta gene repertoires in peripheral lymphocytes from nine sets of identical twins, normal, concordant or discordant for rheumatoid arthritis (RA), were studied. The TcR V beta results were compared with TcR V gamma frequencies in the same specimens as determined by APCR-ELISA and cDNA sequence analysis. The results showed a marked similarity in the TcR V beta gene repertoires between identical twins, compared to unrelated subjects (p < 0.05) whether or not they were concordant or discordant for RA. In contrast, the TcR V gamma gene repertoires in the monozygotic twins differed as much as in controls. The data imply that (a) the human TcR V beta gene repertoire in peripheral blood is genetically controlled, whereas (b) the TcR V gamma gene repertoire is primarily influenced by environmental stimuli, and (c) RA causes no consistent change in TcR V beta repertoire of peripheral blood. The APCR-ELISA method, in the context of large-scale family and population studies, should facilitate a more precise delineation of the genetic factors that regulate human TcR V beta expression.

Lack of selective V beta deletion in peripheral CD4+ T cells of human immunodeficiency virus-infected infants.

To investigate the possibility of super-antigen-mediated deletions of T cells expressing particular T cell receptor V beta (TcR V beta) gene segments during human immunodeficiency virus (HIV) infection, TcR V beta usage in CD4+ and CD8+ subsets was analyzed in a cohort of infants maternally infected by HIV and in a group of healthy neonates. We used a semi-quantitative anchored polymerase chain reaction technique together with cytofluorographic analysis with anti-V beta monoclonal antibodies. The representation of the 24 V beta families in CD4+ and CD8+ T cells from normal neonates was very similar to that in adults. Preferential expression of V beta 2 in the CD4+ subset was observed in both the neonates and in healthy adults. The representation of the 24 V beta families in peripheral CD4+ T cells from the HIV-infected infants showed no selective V beta deletion, even when the CD4+ subset was globally depleted. Moreover, the main characteristics of the control group (predominance of certain V beta families and V beta 2 skewing towards the CD4+ subset) were also present in all the HIV-infected infants.

The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein

Trichohyalin is an intermediate filament-associated protein that associates in regular arrays with keratin intermediate filaments (KIF) of the inner root sheath cells of the hair follicle and the granular layer of the epidermis and is a known substrate of transglutaminases. We have determined the full-length sequence of human trichohyalin by use of RNA-mediated anchored polymerase chain reaction methods and from a genomic clone and analyzed its potential secondary structure. We show here that trichohyalin may have at least three important functions in these cells. The protein of 248 kDa is unusual in that it contains one of the highest contents of charged residues of any protein. Of several defined domains, domains 2-4, 6, and 8 are almost entirely alpha-helical, configured as a series of peptide repeats of varying regularity, and are thought to form a single-stranded alpha-helical rod stabilized by ionic interactions between successive turns of the alpha-helix. Domain 6 is the most regular and may bind KIF directly by ionic interactions. Domains 5 and 7 are less well organized and may introduce folds in the molecule. Thus, human trichohyalin is predicted to be an elongated flexible rod at least 215 nm long and to function as a KIF-associated protein by cross-linking the filaments in loose networks. In addition, trichohyalin is similar to, but several times longer than, involucrin, a known cell envelope constituent, so that together, involucrin and trichohyalin may serve as scaffold proteins in the organization of the cell envelope of these cells or even anchor the cell envelope to the KIF network. Finally, trichohyalin possesses a pair of functional calcium-binding domains of the EF-hand type at its amino terminus that may be involved in its calcium-dependent postsynthetic processing during terminal differentiation.

Characterization of the human T cell receptor α‐chain repertoire and demonstration of a genetic influence on Vα usage

Despite considerable knowledge of the determinants and profile of the T cell receptor (TCR) beta-chain repertoire, very little is known about the repertoire of the V alpha and J alpha segments of the TCR in man or mouse. We have used the anchored polymerase chain reaction (PCR) to analyze the human TCR alpha-chain repertoire at the mRNA level in peripheral blood lymphocytes. We find marked differences in the usage of individual V alpha segments. The V alpha repertoire is determined both by differences in the use of individual V alpha gene segments at the time of gene rearrangement and by genetic influences on the selection of the expressed V alpha repertoire, presumably during thymic maturation. Analysis of the J alpha repertoire shows non-random usage of individual J alpha segments but no obvious relationship to chromosomal position. We also report three new V alpha segments, three new J alpha segments and possible allelic variation in V alpha gene segments. Two unusual TCR alpha transcripts are described which add new information on the nature of TCR alpha transcript assembly.

Restricted kappa chain expression in early ontogeny: biased utilization of V kappa exons and preferential V kappa-J kappa recombinations.

To determine the extent of kappa chain diversity in the preimmune repertoire early in development, kappa cDNA libraries were analyzed from 15-d old fetal omentum, 18-d-old fetal liver, and 3-wk old bone marrow. An anchored polymerase chain reaction approach was used to avoid bias for particular V kappa families. From the sequence analysis of 27 bone marrow clones, 10 different families and 20 unique V kappa genes were identified. In contrast, the V kappa expression in the fetus is highly restricted and clearly differs from the broader distribution see in 3-wk-old bone marrow. Although several V kappa families were represented in the fetal library including V kappa 9, V kappa 10, V kappa 4,5, V kappa 8, and V kappa 1, one or two members of individual families were observed repeatedly. The fetal liver and omentum libraries were found to be largely overlapping. Given the V kappa families/exons identified in the fetal sequences, the mechanism of kappa rearrangements in the early repertoire appears to occur predominantly by inversion. Importantly, the fetal repertoire was further restricted by dominant V kappa-J kappa combinations such as V kappa 4,5-J kappa 5, V kappa 9-J kappa 4, and V kappa 10-J kappa 1. Since in some cases independent rearrangements could be established, the results indicate a bias for particular V kappa-J kappa joins. The results also suggest that clonal expansion/selection in the fetal repertoire takes place after light chain rearrangement as opposed to at the pre-B cell level in the bone marrow. The restriction observed in kappa light chain expression together with known restrictions in gene usage and junctional diversity at the heavy chain level indicate a remarkably conserved fetal repertoire.

Normal T cell receptor V beta usage in a primary immunodeficiency associated with HLA class II deficiency.

The human T cell receptor was studied using an anchored-polymerase chain reaction (A-PCR) and hybridization with V beta-specific oligonucleotide probes, together with the few anti-V beta monoclonal antibodies (mAb) available. After confirming the semiquantitative and reproducible nature of the A-PCR technique, we assessed the complete V beta repertoire in sorted CD4+ and CD8+ lymphocyte populations from three normal donors. These experiments confirmed the absence of V beta-restricted deletions in human peripheral cells, in contrast to several mouse strains. This feature makes it difficult to study negative selection in man, given the apparent absence of an endogenous superantigen corresponding to the Mls system in the mouse. To investigate human V beta repertoire shaping, we studied V beta usage in CD4+ and CD8+ T cells from children with an inherited immunodeficiency characterized by defective expression of human leukocyte antigen class II molecules. An initial study using anti-V beta monoclonal antibodies failed to show significant abnormalities in V beta usage. Four patients analyzed using the A-PCR method all had a polyclonal V beta repertoire, suggesting normal positive selection and raising questions as to the importance of V beta major histocompatibility complex (MHC) interactions and the role of thymic MHC density in shaping the V beta repertoire.

Alternatively spliced T cell receptor transcripts expressed in human T lymphocytes

We used the anchored-polymerase chain reaction (A-PCR) procedure to study human TCR transcripts derived from a variety of polyclonal T cell populations. In this series of experiments, 31 ‘unusual’ cDNAs, which do not include exclusively V-J-C, J-C or 5' C genomic sequences, were identified. Ten of these were found to represent distinct types of alternatively spliced TCR α transcripts whose structure is derived from unusual splicing of one, two or even three intervening intronic sequences. The splicing events led to either conservation of a novel exon in the mRNA structure (designated aE1α-aE5α) between the V-J and C segments or to deletion of the 3'V region-J segment. In three cases, the alternatively spliced exons (aE1α-aE3α) interrupt the open translational reading frame of the corresponding V-Jα segment. Nineteen and two cDNA represent sterile Cβ or Cδ transcripts, respectively. Their structures are derived from the conservation of a non-translatable exon, aE1β or aE1δ, which is precisely spliced at the 5' end of the corresponding C exon sequences. Interestingly, the 3' region of the aE1β sequence is homologous to the murine Cβ0 exon. Together, these results led to the characterization of nine novel exons in the TCR α, β and δ loci.

Divergent T cell receptor gamma repertoires in rheumatoid arthritis monozygotic twins.

To determine if the expressed T cell receptor (TCR) gamma repertoire is altered in rheumatoid arthritis (RA). Peripheral blood lymphocytes were collected from monozygotic twins who were either concordant or discordant for RA, or from a normal twin pair. TCR gamma-specific complementary DNA libraries were constructed using the anchored polymerase chain reaction. Gene usage was analyzed by plaque hybridization and sequencing. The expressed TCR V gamma repertoires both in RA patients and normal subjects were extremely diverse. Monozygotic twins who were concordant for RA expressed very different frequencies of TCR V gamma genes. RA does not lead to a specific clonal expansion or deletion of TCR V gamma genes in peripheral blood.

Autoaggressive myocytotoxic T lymphocytes expressing an unusual gamma/delta T cell receptor.

Polymyositis mediated by gamma/delta T cells is a unique disease in which autoaggressive T lymphocytes surround, invade, and destroy muscle fibers. Histochemically, the vast majority of muscle-infiltrating T cells in a patient with polymyositis were reactive with a pan-gamma/delta T cell receptor (TCR)-specific monoclonal antibody (TCR-delta 1+), but unlike > 90% of peripheral blood gamma/delta T cells, these lymphocytes did not react with V delta 1- or V gamma 9-specific antibodies (A13- and Ti gamma A-, respectively). Differential reactivity with two different V delta 2-specific monoclonal antibodies (BB3-/TiV-delta 2+) indicated that the infiltrating T cells express a V delta 2-containing TCR with unusual additional structural features. Using conventional and anchored polymerase chain reaction for the analysis of TCR transcripts, we found a striking predominance of one unusual V delta 2-J delta 3 recombination and one V gamma 3-J gamma 1 recombination. Both the unusual phenotype (TCR-delta 1+/A13-/Ti gamma A-/BB3-/TiV-delta 2+) and the dominance of distinct TCR transcripts are compatible with the assumption that one T cell clone, which expresses a V gamma 3-J gamma 1-C gamma 2/V delta 2-J delta 3-C delta disulfide-linked TCR, dominates among the infiltrating T cells of the polymyositis muscle specimen analyzed.

Cloning of the gene coding for a human receptor for formyl peptides. Characterization of a Promoter Region and Evidence for Polymorphic Expression

Recently we reported that, in HL-60 cells, transcription of the formyl peptide receptor (FPR) gene can be up- and downregulated by agents that induce differentiation of HL-60 cells into neutrophils. To begin studying the mechanisms involved in regulation of FPR gene expression, we cloned two human cDNAs and the gene coding for FPR. The genomic clone (pINF14) contained a 14.5-kb insert. A 2.7-kb EcoRI fragment was obtained from pINF14 that hybridized with an FPR open reading frame probe. The EcoRI fragment was sequenced and found to contain an intronless FPR open reading frame. Sequence alignment of the EcoRI genomic fragment with the FPR cDNA revealed that the first 31 bases of 5' untranslated FPR cDNA were not represented in the genomic fragment. Furthermore, a splicing consensus sequence was present in the genomic fragment at the site of divergence with the cDNA sequence. Restriction mapping and Southern blot analysis identified a 121-bp fragment that contained the sequence corresponding to the first 31 bases of 5' untranslated FPR cDNA. An additional (previously undescribed) 15-bp cDNA sequence in the 5' end of FPR were identified using an anchored polymerase chain reaction. This sequence was also contained in the genomic 121-bp fragment. This 121-bp fragment was located 5.2 kb (intron) upstream of the FPR open reading frame. It contained an unusual TATA box and displayed transcriptional activity in vitro and in vivo. Potential binding sites for AP-1 and glucocorticoid receptor were identified upstream of the putative TATA box.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning of the porcine Calbindin-D9k complementary deoxyribonucleic acid by anchored polymerase chain reaction technique.

The Calbindin-D9k (CaBP-9k) is a cytosolic calcium binding protein expressed in the mammalian intestine, placenta, and uterus. The protein is probably involved in calcium transport across the intestinal and placental epithelia. In uterus, a function in controlling myometrial activity involving intracellular calcium has been postulated. The amino acid sequence of the porcine CaBP-9k has been determined from intestine. The cDNAs for the bovine, murine, and rat CaBP-9k have been cloned. The objective of this study was the cloning of the porcine cDNA encoding the CaBP-9k. We performed the anchored polymerase chain reaction (PCR) technique using rat and bovine cDNA sequence-derived primers for amplification of intestinal cDNA. Both 5' and 3' amplification products were cloned and sequenced. The sequences revealed the full-length cDNA encoding the porcine CaBP-9k, coding region for 79 amino acids, 57 nucleotides 5' and 149 nucleotides 3' noncoding region. The inferred amino acid sequence is identical to the published amino acid sequence, except for one residue. The porcine CaBP-9k cDNA is 82.8% and 69.1% homologous with the bovine and rat sequences, respectively. Both bovine and porcine cDNAs contain a stretch of approximately 50 nucleotides not found in the rat sequence. Northern analysis showed a 600 nucleotide transcript in intestine, kidney, and uterus.

The human T cell receptor gamma genes are transcribed from tata-less promoters containing a conserved heptamer sequence

We have used the anchored polymerase chain reaction (A-PCR) to clone and compare the 5' upstream regions of the human T cell receptor gamma (TRG) genes. Whereas little homology was found among subgroups I, II, III and IV, sequence alignment of TRG subgroup I members revealed a high degree of homology in the 5' sequences. A conserved heptamer sequence (CTGCAGG), which was found upstream from the translation intiation site of all TRG genes in our analysis. Determination of the transcription initiation site located the conserved heptamer 65 base pairs upstream from the cap sites of V5. No TATA box or other cis -acting promoter sequences could be identified in any of the human TRG upstream sequences.

Identification of novel human T-cell receptor V beta gene segments by the anchored-polymerase chain reaction.

We have studied the diversity of the expressed human T-cell receptor (TCR) beta-chain repertoire by analysis of mRNA from unstimulated peripheral blood T-cells. The anchored-polymerase chain reaction (PCR) was used to isolate TCRB transcripts. Of 20 full or near full-length functional transcripts sequenced, two were novel TCRVB gene segments. They have strong sequence similarities to the known TCRBV5, and 8 subfamilies. Southern blot analysis and sequence-specific oligonucleotide hybridization confirmed: a) that these sequences are present in genomic DNA; b) their relationship to the known TCRBV families. A TCRBV sequence similar to a recently identified novel TCRBV24 subfamily was also found. We show by southern blotting that this sequence forms a single member subfamily, and by deletion analysis of T-cell lines, we have mapped this sequence to lie between the genes which encode the TCRBV8.1 and TCRBV5.3 gene segments. The results show that the anchored PCR is a powerful tool in the analysis of the TCR repertoire, which may contain more V gene segments than previously defined.

Cloning and mapping of 5′ exons from the gene encoding chicken beta nerve growth factor

An NGF cDNA containing the 5′ exons of the nerve growth factor (NGF) messenger was obtained from chicken heart mRNA using the anchored polymerase chain reaction technique. Alignment of the chicken with the corresponding murine and human sequences reveals interspecies similarities. A sequence corresponding to an exon found only in the NGF messenger, which is abundant in the submaxillary gland of the male mouse, is present in the chicken NGF cDNA. The first non-coding exons of the NGF gene are much less conserved between chicken and mouse or human than the region of the last exon encoding the mature protein. After the cloning of the chicken NGF gene from a cosmid library, the chicken NGF exons have been located within 20 kb of DNA. The chicken NGF gene is therefore shorter than its murine counterpart which spans more than 43 kb. Furthermore, the organization of the chicken and murine NGF genes markedly differs in their 5′ portion.

Characterization of the rat pulmonary surfactant protein A promoter.

The expression of the pulmonary surfactant protein A (SP-A) is developmentally regulated and controlled by several hormones. In an attempt to characterize cis-acting elements involved in the regulation of SP-A expression, we have cloned the 5' flanking sequence of the rat SP-A gene. The promoter region contains a TATA box but no CAAT box. The transcription start site has been identified by anchored polymerase chain reaction and S1 nuclease mapping of the mature and precursor transcripts. S1 mapping of precursor transcripts has confirmed the stimulating effect of glucocorticoids on SP-A rat gene transcription in vivo. This hormonal effect may be mediated by a putative glucocorticoid responsive element located 140 bp upstream from the initiation site and protected against DNase 1 digestion in footprinting experiments. In vitro transcription of a G-free reporter cassette linked to the 212-bp 5' flanking DNA fragment has established that this putative promoter region is functional. Efficient transcription of the G-free reporter cassette was obtained with cell-free fetal lung extracts, whereas no transcript was detectable with cell-free liver extracts. Comparative analysis of the human and rat 5' flanking sequences shows the presence of strongly conserved motifs, unrelated to previously known consensus sequences. Some of these motifs, specifically protected in DNase 1 footprinting studies, could therefore be involved in the regulation of SP-A gene expression.

Diversity of novel recombining elements suggests developmentally programmed expression of the T cell receptor alpha/delta locus.

During fetal ontogeny, the first wave of gamma delta T lymphocytes appears in the thymus at day 14 of gestation assembling predominantly T cell receptors (TcR) with V gamma 3 and V delta 1. To identify V delta gene segments that are transcribed at day 16, subsequent to the first wave of V delta 1 expression, delta chain cDNA was amplified by the anchored polymerase chain reaction with single-sided specificity for C delta. Unexpectedly, most of the cDNA clones do not contain V gene segments. In some cDNA clones an alternative splice from the leader exon to the C delta exon has deleted the whole variable region exon. In other cDNA clones, multiple non-V-like elements are juxtaposed to the D delta 2 and J delta 1 gene segments. A large number of these diverse elements appear to be rearranged in fetal thymocytes, bringing V alpha gene segments located upstream of the recombining element into proximity to the J alpha locus. It is proposed that these rearrangements make irreversible the commitment to the TcR alpha beta lineage and determine a programmed read out of different clusters of V alpha gene segments.