PubMed Health⌕ Search

Biomedical subjects

A J Feeney

Publications and source records attributed to A J Feeney.

At least 19 recordsLinked to original sources
PubMed1998

Decreased frequency of rearrangement due to the synergistic effect of nucleotide changes in the heptamer and nonamer of the recombination signal sequence of the V kappa gene A2b, which is associated with increased susceptibility of Navajos to Haemophilus influenzae type b disease.

Navajos and genetically related populations have a 10-fold increased incidence of Haemophilus influenzae type b (Hib) disease compared with control populations. The Vkappa gene A2 is used to encode the majority of anti-Hib Abs, and these are the highest affinity anti-Hib Abs. Navajos carry a different allele of the A2 gene segment (A2b) that is defective in its ability to undergo V-J recombination. The A2b allele has only three nucleotide changes from the commonly occurring A2a allele, two of which could potentially affect its ability to recombine. In this study we used two independent in vitro assays to test whether the nucleotide change found in the A2b promoter and/or in the A2b recombination signal sequence (RSS) might be responsible for the decrease in recombination frequency observed in vivo. Using a luciferase reporter gene assay, we found no significant difference between A2a and A2b promoter activities. However, the competition recombination substrate assay showed a 4.5-fold reduction in the relative frequency of recombination of the A2b RSS compared with A2a. We show that this decreased frequency is due to a synergistic effect of the unique nucleotide change present in the heptamer of the A2b RSS and the shared nucleotide change present in the nonamer of both A2b and A2a. This in vitro relative frequency of rearrangement is not significantly different from that observed in vivo; therefore, the A2b RSS is probably the factor associated with the increased susceptibility to Hib disease among individuals carrying the A2b allele.

Alleles↗
PubMed1998

Sequence of the spacer in the recombination signal sequence affects V(D)J rearrangement frequency and correlates with nonrandom Vkappa usage in vivo.

Functional variable (V), diversity (D), and joining (J) gene segments contribute unequally to the primary repertoire. One factor contributing to this nonrandom usage is the relative frequency with which the different gene segments rearrange. Variation from the consensus sequence in the heptamer and nonamer of the recombination signal sequence (RSS) is therefore considered a major factor affecting the relative representation of gene segments in the primary repertoire. In this study, we show that the sequence of the spacer is also a determinant factor contributing to the frequency of rearrangement. Moreover, the effect of the spacer on recombination rates of various human Vkappa gene segments in vitro correlates with their frequency of rearrangement in vivo in pre-B cells and with their representation in the peripheral repertoire.

B-Lymphocytes↗
PubMed1998

V(H) replacement is unlikely to contribute significantly to receptor editing due to an ineffectual embedded recombination signal sequence.

Receptor editing is a process consisting of replacement of pre-existing H or L chain rearrangements by secondary rearrangements. This process could serve to remove autoreactive specificities, or to rescue loci with non-functional rearrangements. At the H chain locus, functional replacement of a V(H)DJ(H) rearrangement by an upstream V(H) requires the presence of an embedded RSS located in reverse orientation near the 3' end of the V(H) segment. Although most V(H) genes contain a fairly consensus embedded heptamer, the nonamer sequence bears little resemblance to the consensus RSS nonamer. Therefore, the physiologic rate of H chain editing by V(H) replacement is yet unknown. In this study, we used both conventional and sensitive competition recombination substrate assays to determine the recombination frequency of the V(H)1X embedded RSS relative to consensus and non-consensus RSS's. Results show no detectable recombination of the 81X embedded RSS in a recombination substrate, and the competition substrate allows us to estimate that the 81X embedded RSS recombines at least 1300 fold less often than a consensus RSS. This suggests that V(H) gene replacement is not responsible for the decrease in representation of the 81X gene during differentiation. Furthermore, since the sequence of the embedded RSS is very similar for many V(H) genes, our results suggest that receptor editing of the H chain will be an infrequent event, leaving L chain editing as the main mode of avoiding autoreactive specificities in vivo.

Gene Rearrangement, B-Lymphocyte, Heavy Chain↗
PubMed1997

Human cord blood kappa repertoire.

To determine the Vkappa gene utilization in cord blood, we made libraries of Igkappa sequences from two cord blood cDNA samples. The rearranged sequences were amplified using random amplification of cDNA ends PCR, ensuring unbiased amplification of all Vkappa genes. Although the human kappa locus contains approximately 38 potentially functional V genes, we observed that approximately 75% of the 146 sequences from our two samples used only nine Vkappa genes. Using leader-specific primers, we also amplified VkappaI and VkappaIII rearrangements from genomic DNA from one of these individuals. Nonproductive rearrangements give an approximation of the relative frequency of gene rearrangement. Some of the genes that were overused in the cDNA libraries were also observed to rearrange frequently, but others did not show high rearrangement frequencies, suggesting that cellular selection caused their increase in the periphery. Surprisingly, we observed a high frequency of rearrangements using L9, which has been reported to be a defective Vkappa gene. Sequence analysis of the unrearranged gene revealed two new functional alleles of this gene. We observed that N nucleotides were present in 29% of the productive sequences from cord blood DNA and RNA. To determine the actual rate of N region addition, we analyzed V-J junctions of rearrangements of two nonfunctional V genes. Forty-six percent of those cord blood sequences contained N regions. In comparison, 57% of junctions of the rearranged nonfunctional gene from adult PBMC contained N regions. Finally, we observed that CDR3 length heterogeneity was more pronounced for VkappaIII genes than for all of the other Vkappa families.

Adult↗
PubMed1997

Both E12 and E47 allow commitment to the B cell lineage.

The E2A gene products, E12 and E47, are required for proper B cell development. Mice lacking the E2A gene products generate only a very small number of B220+ cells, which lack immunoglobulin DJ(H) rearrangements. We have now generated mice expressing either E12 or E47. B cell development in mice expressing E12 but lacking E47 is perturbed at the pro-B cell stage, and these mice lack IgM+B220+ B cells in both bone marrow and spleen. IgM+B220+ B cells can be detected, albeit at significantly reduced levels, in the bone marrow and spleen of mice lacking E12. Ectopic expression of both E12 and E47 in a null mutant background shows that E12 and E47 act in concert to promote B lineage development. Taken together, the data indicate that both E12 and E47 allow commitment to the B cell lineage and act synergistically to promote B lymphocyte maturation.

Animals↗
PubMed1997

Nucleotide deletion and P addition in V(D)J recombination: a determinant role of the coding-end sequence.

During V(D)J recombination, the coding ends to be joined are extensively modified. Those modifications, termed coding-end processing, consist of removal and addition of various numbers of nucleotides. We previously showed in vivo that coding-end processing is specific for each coding end, suggesting that specific motifs in a coding-end sequence influence nucleotide deletion and P-region formation. In this study, we created a panel of recombination substrates containing actual immunoglobulin and T-cell receptor coding-end sequences and dissected the role of each motif by comparing its processing pattern with those of variants containing minimal nucleotide changes from the original sequence. Our results demonstrate the determinant role of specific sequence motifs on coding-end processing and also the importance of the context in which they are found. We show that minimal nucleotide changes in key positions of a coding-end sequence can result in dramatic changes in the processing pattern. We propose that each coding-end sequence dictates a unique hairpin structure, the result of a particular energy conformation between nucleotides organizing the loop and the stem, and that the interplay between this structure and specific sequence motifs influences the frequency and location of nicks which open the coding-end hairpin. These findings indicate that the sequences of the coding ends determine their own processing and have a profound impact on the development of the primary B- and T-cell repertoires.

Animals↗
PubMed1996

Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities.

PU.1 is a member of the ets family of transcription factors and is expressed exclusively in cells of the hematopoietic lineage. Mice homozygous for a disruption in the PU.1 DNA binding domain are born alive but die of severe septicemia within 48 h. The analysis of these neonates revealed a lack of mature macrophages, neutrophils, B cells and T cells, although erythrocytes and megakaryocytes were present. The absence of lymphoid commitment and development in null mice was not absolute, since mice maintained on antibiotics began to develop normal appearing T cells 3-5 days after birth. In contrast, mature B cells remained undetectable in these older mice. Within the myeloid lineage, despite a lack of macrophages in the older antibiotic-treated animals, a few cells with the characteristics of neutrophils began to appear by day 3. While the PU.1 protein appears not to be essential for myeloid and lymphoid lineage commitment, it is absolutely required for the normal differentiation of B cells and macrophages.

Animals↗
PubMed1996

A defective Vkappa A2 allele in Navajos which may play a role in increased susceptibility to haemophilus influenzae type b disease.

The antibody response to H. influenzae type b (Hib) is pauciclonal, and is dominated by antibodies using the VkappaA2 gene. Navajos have a 5-10-fold increased incidence of Hib disease compared with control populations. We hypothesized that a polymorphism in one of the genes in this oligoclonal response may lead to increased disease susceptibility. Since the predominant A2+ anti-Hib antibodies have high avidity for Hib and can be unmutated, the A2 Vkappa gene was analyzed. Over half of the Navajos studied, but only one control individual, had a new allele of A2, termed A2b, with three changes from the published A2 germline sequence. One of the changes was in the recombination signal sequence, suggesting that the A2b allele might not undergo V-J rearrangement very frequently. This possibility was confirmed by analyzing the relative frequency of non-productive A2 rearrangements in A2a/b heterozygous Navajos. Many fewer A2b rearrangements were observed, showing that the A2b allele is defective in its ability to undergo rearrangement. The prevalence of this allele in Navajos may play a role in their increased susceptibility to invasive Hib disease. If so, it would underscore the importance of the germline Ig repertoire for protective antibody responses to pathogenic bacteria in unimmunized children.

Alleles↗
PubMed1996

New alleles of IGKV genes A2 and A18 suggest significant human IGKV locus polymorphism.

The human kappa light chain consists of approximately 35 potentially functional IGKV genes. However, an estimation of the diversity in the IGKV repertoire of an individual will be affected by the extent of polymorphisms for the different IGKV genes and their patterns of inheritance. To date, little information is available to indicate the extent of allelic variation of the IGKV genes. We examined the extent of allelism for one IGKV gene pair, the distal region A2 gene and its closely related proximal region duplicate A18. We found two new alleles for A2 and one new allele for A18, and sequenced approximately 1 kilobase flanking each gene. The new A18 allele, unlike the originally described allele, appears to be functional. All these alleles were found at relatively high frequencies in the four ethnic populations studied, with the exception of the defective A2b allele which was highly represented only in Navajos. The originally described A2a allele encodes for the predominant protective antibody against Haemophilus influenzae. Therefore, the patterns of allelic inheritance described for this IGKV gene pair indicate that allelism in the IGKV locus is likely to have a significant impact on immune responses.

Alleles↗
PubMed1995

Influence of coding-end sequence on coding-end processing in V(D)J recombination.

The large diversity of the Ig and TCR repertoires is accounted for by combinatorial assembly of the germ-line-encoded V, D, and J gene segments, as well as extensive modification at the junctions during the recombination process. Those modifications, termed coding-end processing, consist of removal and addition of an apparently random number of nucleotides. To obtain further insights into the mechanism of the coding-end processing, we constructed a large data base of several Ig and TCR coding ends obtained in vivo, using conditions that avoid potential bias by cellular selection events. We show that the processing patterns are not random, but rather specific for each coding end, suggesting that specific motifs in the coding-end sequence influence the processing. We found a good correlation between the presence of internal stretches of at least three A.T nucleotides, absence of stretches of G.C nucleotides, and high average nucleotide deletion. Based on a detailed analysis of the processing patterns, we propose that nicks of the hairpin intermediate take place preferentially in potential open structures formed by weaker pairings of A.T stretches. Together, these findings indicate that the sequence of the coding end plays an important role in nonrandom aspects of the recombination mechanism. This suggests that coding-end sequences might have been selected throughout evolution to participate in an early control of the development of the primary repertoire.

Animals↗
PubMed1995

Coding end processing is similar throughout ontogeny.

During the recombination process, extensive processing of the coding ends provides tremendous potential diversity to the joint of any two gene segments. However, the diversity of the newborn B and T cell repertoires is greatly reduced compared with that of the adult. At the mechanistic level, this difference is primarily due to the absence of terminal deoxynucleotidyltransferase expression until the first week after birth. Additionally, one direct consequence of the lack of N regions early in ontogeny is the more frequent occurrence of homology-directed recombination, reducing even further the potential of diversity. Other enzymatic factors could also contribute to this ontogenic difference. However, the use of the homology-directed recombination pathway early in life obscures the analysis of the coding end processing. In this study we compared the coding end processing throughout ontogeny, in normal and terminal deoxynucleotidyltransferase -/- mice in the presence of minimal homology-directed recombination. The analysis of partial D-J joints allowed us to avoid potential bias by early selection events. Our results show that the extent of nucleotide deletion of a given end is consistent throughout ontogeny in the presence or absence of terminal deoxynucleotidyltransferase. However, a distinctive processing pattern is observed for each coding end.

Age Factors↗
PubMed1995

Analysis of homology-directed recombination in VDJ junctions from cytoplasmic Ig- pre-B cells of newborn mice.

We previously showed that most VD and DJ gene combinations from newborn surface Ig- pre-B cells had one to three predominant junctions, all of which occurred at the sites of short sequence homologies between the two coding ends. Because the majority of sequences that are present in pre-B cells are in-frame, however, the possibility existed that the frequency of occurrence of predominant IgH junctions was skewed by proliferation of pre-B cells with productive rearrangements. In this study, we analyzed cytoplasmic Ig- pre-B cells, because these cells should not yet be subject to such selection. Two-thirds of the rearrangements from this population in the adult were out-of-frame, suggesting that these rearrangements are unbiased. In newborn cIg- pre-B cells, DJ junctions still showed the same predominant sequences as sIg- pre-B cells, but there was less use of predominant junctions in VD junctions for three of four different VH genes analyzed. For those three VH genes, an average of 30% of the sequences were in-frame. When only the in-frame rearrangements from these cIg- newborn cells were analyzed, frequencies of predominant VD junctions were comparable to those in sIg- pre-B cells. For sequences using the VHS107/V11 gene, however, 67% of the junctions were created at the site of the same dinucleotide in the V gene, and as a result, 73% of the sequences were in-frame. Thus homology-directed recombination does not initially produce as much junctional homogeneity as anticipated in all VD combinations, although it is a frequently used mechanism in the early fetal/neonatal gene rearrangements.

Animals↗
PubMed1994

E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements.

E12 and E47 are two helix-loop-helix transcription factors that arise by alternative splicing of the E2A gene. Both have been implicated in the regulation of immunoglobulin gene expression. We have now generated E2A (-/-) mice by gene targeting. E2A-null mutant mice fail to generate mature B cells. The arrest of B cell development occurs at an early stage, since no immunoglobulin DJ rearrangements can be detected in homozygous mutant mice. While immunoglobulin germline I mu RAG-1, mb-1, CD19, and lambda 5 transcripts are dramatically reduced in fetal livers of E2A (-/-) mice, B29 and mu degrees transcripts are present, but at lower levels. In addition, we show that Pax-5 transcripts are significantly reduced in fetal livers of E2A (-/-) mice. These data suggest a crucial role for E2A products as central regulators in early B cell differentiation.

Animals↗
PubMed1994

Limited junctional diversity in kappa light chains. Junctional sequences from CD43+B220+ early B cell progenitors resemble those from peripheral B cells.

Among all adult T and B cell Ag receptor chains, only Ig light chains lack N regions. It is thought that this is due to the fact that light chain genes rearrange after heavy chain genes, and that terminal deoxynucleotidyl transferase, the enzyme that adds N regions, is not longer expressed at that stage. However, this concept has been challenged recently by the demonstration that 3 to 10% of B cell precursors (CD43+B220+) appear to rearrange their light chains at approximately the same time as they undergo VH-->DJ rearrangements. To examine N region addition in B cell precursors undergoing early kappa-chain rearrangement, we PCR amplified rearranged V kappa 21 genes from the CD43+B220+ bone marrow cells and compared them to sequences obtained from whole bone marrow and spleen. Unexpectedly, all three populations showed approximately 10% N region containing junctions, most consisting of only one N nucleotide. Thus, even the B cell precursors that rearrange light chains at this early stage of development lack much N region diversity. Twelve percent of the sequences unambiguously contained P regions, which were from 1 to 5 nucleotides in length. All but 2 of the 41 productive rearrangements had the commonly observed CDR3 length of nine amino acids. Many (71%) of the sequences were out of frame. CDR3 length was very restricted in nonproductive rearrangements too, and deletion of nucleotides from V kappa and J kappa gene segments was limited. Thus, even at the level of nonproductive rearrangements, junctional diversity is minimal for kappa-chains.

Animals↗
PubMed1994

Influence of the V(D)J recombination mechanism on the formation of the primary T and B cell repertoires.

T and B cells exploit the mechanism of V(D)J recombination to make diverse or very restricted repertoires at varying times during ontogeny. Fetal repertoires are limited since there are no N nucleotides. Also, if short sequence homologies are present near the coding ends, junctions are preferentially made at that site. For gamma delta TCR, and to a lesser extent for Ig, this results in a very homogeneous population of junctions early in ontogeny. alpha beta TCR, however, have a paucity of homologous stretches, and maintain junctional diversity in the newborn. In both newborns and adults, some coding ends show very restricted nucleotide deletion, while others show heterogeneous and extensive deletion. It appears that the sequences of the coding ends have been selected through evolution as a mechanism to control repertoire formation.

Animals↗
PubMed1994

VH11 bias and normal V-D-J junctions in SCID B lymphocytes rescued by neonatal T cell transfer.

The scid mutation interferes with normal rearrangement of antigen receptor genes, leading to an absence of T and B lymphocytes in most SCID mice. However, the SCID phenotype is "leaky", with an age- and strain-dependent increase in the incidence of mice with small number of T and B cells and readily detectable serum immunoglobulin. Introduction of neonatal T cells into young SCID mice results in a 100% incidence of the leaky phenotype. We have identified the location of antibody secreting cells in T cell-induced leaky SCID mice as the spleen and peritoneal cavity, and we have sequenced 35 productively rearranged immunoglobulin genes from these sites to determine if normal V-D-J recombination was occurring. VH11 sequences with potential autoreactivity were observed frequently in both the peritoneal cavity and spleen of T cell-induced leaky SCID mice, and these sequences were indistinguishable from those recovered from peritoneal cavity B cells from normal C.B-17 mice. Non-VH11 SCID sequences showed fewer N nucleotides and slightly more P nucleotides than normal V-D-J sequences. Many SCID junctions occurred at the site of short sequence homologies. These results suggest that successful V-D-J recombination is occurring with low frequency in all SCID mice, and that neonatal T cell transfer plus autoantigen stimulation allows the long term survival of these B cells.

Animals↗