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E C McKinney

Publications and source records attributed to E C McKinney.

At least 19 recordsLinked to original sources
PubMed2002

The development of primary and secondary lymphoid tissues in the nurse shark Ginglymostoma cirratum: B-cell zones precede dendritic cell immigration and T-cell zone formation during ontogeny of the spleen.

Secondary lymphoid tissue and immunoglobulin (Ig) production in mammals is not fully developed at birth, requiring time postnatally to attain all features required for adaptive immune responses. The immune system of newborn sharks - the oldest vertebrate group having adaptive immunity - also displays immature characteristics such as low serum IgM concentration and high levels of IgM1gj, an innate-like Ig. Primary and secondary lymphoid tissues in sharks and other cartilaginous fish were identified previously, but their cellular organization was not examined in detail. In this study of nurse shark lymphoid tissue, we demonstrate that the adult spleen contains well-defined, highly vascularized white pulp (WP) areas, composed of a central T-cell zone containing a major histocompatibility complex (MHC) class II+ dendritic cell (DC) network and a small number of Ig+ secretory cells, surrounded by smaller zones of surface Ig+ (sIg+) B cells. In neonates, splenic WPs are exclusively B-cell zones containing sIgM+-MHC class IIlow B cells; thus compartmentalized areas with T cells and DCs, as well as surface Ig novel antigen receptor (sIgNAR)-expressing B cells are absent at birth. Not until the pups are 5 months old do these WP areas become adult-like; concomitantly, sIgNAR+ B cells are readily detectable, indicating that this Ig class requires a 'mature immune-responsive environment'. The epigonal organ is the major site of neonatal B lymphopoiesis, based on the presence of developing B cells and recombination-activating gene 1 (RAG1)/terminal deoxynucleotidyl transferase (TdT) expression, indicative of antigen receptor rearrangement; such expression persists into adult life, whereas the spleen has negligible lymphopoietic activity. In adults but not neonates, many secretory B cells reside in the epigonal organ, suggesting, like in mammals, that B cells home to this primary lymphoid tissue after activation in other areas of the body.

Animals↗
PubMed2001

A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG.

In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM(1gj), from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells ("germline-joined"). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H(1gj) in primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H(1gj) chain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM(1gj). Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system.

Amino Acid Sequence↗
PubMed2001

One plant actin isovariant, ACT7, is induced by auxin and required for normal callus formation.

During plant growth and development, the phytohormone auxin induces a wide array of changes that include cell division, cell expansion, cell differentiation, and organ initiation. It has been suggested that the actin cytoskeleton plays an active role in the elaboration of these responses by directing specific changes in cell morphology and cytoarchitecture. Here we demonstrate that the promoter and the protein product of one of the Arabidopsis vegetative actin genes, ACT7, are rapidly and strongly induced in response to exogenous auxin in the cultured tissues of Arabidopsis. Homozygous act7-1 mutant plants were slow to produce callus tissue in response to hormones, and the mutant callus contained at least two to three times lower levels of ACT7 protein than did the wild-type callus. On the other hand, a null mutation in ACT2, another vegetative actin gene, did not significantly affect callus formation from leaf or root tissue. Complementation of the act7-1 mutants with the ACT7 genomic sequence restored their ability to produce callus at rates similar to those of wild-type plants, confirming that the ACT7 gene is required for callus formation. Immunolabeling of callus tissue with actin subclass-specific antibodies revealed that the predominant ACT7 is coexpressed with the other actin proteins. We suggest that the coexpression, and probably the copolymerization, of the abundant ACT7 with the other actin isovariants in cultured cells may facilitate isovariant dynamics well suited for cellular responses to external stimuli such as hormones.

Actins↗
PubMed2001

Small changes in the regulation of one Arabidopsis profilin isovariant, PRF1, alter seedling development.

Profilin (PRF) is a low-molecular-weight actin binding protein encoded by a diverse gene family in plants. Arabidopsis PRF1 transcripts are moderately well expressed in all vegetative organs. A regulatory mutant in PRF1, prf1-1, was isolated from a library of T-DNA insertions. The insertion disrupted the promoter region of PRF1 100 bp upstream from the transcriptional start site. Although steady state levels of PRF1 transcripts appeared normal in mature prf1-1 plants, the levels in young seedlings were only one-half those observed in wild type. Reactions with a PRF1 isovariant-specific monoclonal antiserum and general anti-profilin antisera demonstrated that PRF1 protein levels also were one-half those found in wild-type seedlings, although total profilin levels were unaffected. Mutant seedlings no longer could downregulate PRF1 levels in the light, as did wild type. Consistent with their molecular phenotypes, young mutant seedlings displayed several morphological phenotypes but developed into apparently normal adult plants. Their initial germination rate and development were slow, and they produced excessive numbers of root hairs. Mutant seedlings had abnormally raised cotyledons, elongated hypocotyls, and elongated cells in the hypocotyl, typical of phenotypes associated with some defects in light and circadian responses. A wild-type PRF1 transgene fully complements the hypocotyl phenotypes in the prf1-1 mutant. The ability of profilin to regulate actin polymerization and participate directly in signal transduction pathways is discussed in light of the prf1-1 phenotypes.

Arabidopsis↗
PubMed2000

Primitive synteny of vertebrate major histocompatibility complex class I and class II genes.

Major histocompatibility complex (MHC) class I and class II molecules bind to and display peptidic antigens acquired from pathogens that are recognized by lymphocytes coordinating and executing adaptive immune responses. The two classes of MHC proteins have nearly identical tertiary structures and were derived from a common ancestor that probably existed not long before the emergence of the cartilaginous fish. Class I and class II genes are genetically linked in tetrapods but are not syntenic in teleost fish, a phylogenetic taxon derived from the oldest vertebrate ancestor examined to date. Cartilaginous fish (sharks, skates, and rays) are in the oldest taxon of extant jawed vertebrates; we have carried out segregation analyses in two families of nurse sharks and one family of the banded houndshark that revealed a close linkage of class IIalpha and beta genes both with each other and with the classical class I (class Ia) gene. These results strongly suggest that the primordial duplication giving rise to classical class I and class II occurred in cis, and the close linkage between these two classes of genes has been maintained for at least 460 million years in representatives of most vertebrate taxa.

Amino Acid Sequence↗
PubMed1999

The evolution of new structures: clues from plant cytoskeletal genes.

How large numbers of genes were recruited simultaneously to build new organ structures is one of the greatest puzzles in evolutionary biology. Here, we present data suggesting that the vegetative and reproductive classes of actins and other cytoskeletal proteins arose concurrently with the macroevolutionary divergence of leaves and reproductive structures in the earliest land plants. That the cytoskeleton is essential for physically programming the development of organs and tissues is well established. Thus, we propose that this regulatory dichotomy represents an ancient landmark event in the global regulation of hundreds of higher-plant genes, an event that is linked to the macroevolution of plant vegetative and reproductive organs. The recent availability of sequence and expression data for large numbers of plant genes should make it possible to dissect this and other major macroevolutionary events.

Actins↗
PubMed1999

The late pollen-specific actins in angiosperms.

The actin gene family of Arabidopsis has eight functional genes that are grouped into two ancient classes, vegetative and reproductive, and into five subclasses based on their phylogeny and mRNA expression patterns. Progress in deciphering the functional significance of this diversity is hindered by the lack of tools that can distinguish the highly conserved subclasses of actin proteins at the biochemical and cellular level. In order to address the functional diversity of actin isovariants, we have used Arabidopsis recombinant actins as immunogens and produced several new anti-actin monoclonal antibodies. One of them, MAb45a, specifically recognizes two closely related reproductive subclasses of actins. On immunoblots, MAb45a reacts strongly with actins expressed in mature pollen but not with actins in other Arabidopsis tissues. Moreover, immunocytochemical studies show that this antibody can distinguish actin filaments in pollen tubes from those in most vegetative tissues. Peptide competition analyses demonstrate that asparagine at position 79 (Asn79) within an otherwise conserved sequence is essential for MAb45a specificity. Actins with the Asn79 epitope are also expressed in the mature pollen from diverse angiosperms and Ephedra but not from lower gymnosperms, suggesting that this epitope arose in an ancestor common to angiosperms and advanced gymnosperms more than 220 million years ago. During late pollen development in angio- sperms there is a switch in expression of actins from vegetative to predominantly reproductive subclasses, perhaps to fulfil the unique functions of pollen in fertilization.

Actins↗
PubMed1998

Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins.

We recently have identified an antigen receptor in sharks called NAR (new or nurse shark antigen receptor) that is secreted by splenocytes but does not associate with Ig light (L) chains. The NAR variable (V) region undergoes high levels of somatic mutation and is equally divergent from both Ig and T cell receptors (TCR). Here we show by electron microscopy that NAR V regions, unlike those of conventional Ig and TCR, do not form dimers but rather are independent, flexible domains. This unusual feature is analogous to bona fide camelid IgG in which modifications of Ig heavy chain V (VH) sequences prevent dimer formation with L chains. NAR also displays a uniquely flexible constant (C) region. Sequence analysis and modeling show that there are only two types of expressed NAR genes, each having different combinations of noncanonical cysteine (Cys) residues in the V domains that likely form disulfide bonds to stabilize the single antigen-recognition unit. In one NAR class, rearrangement events result in mature genes encoding an even number of Cys (two or four) in complementarity-determining region 3 (CDR3), which is analogous to Cys codon expression in an unusual human diversity (D) segment family. The NAR CDR3 Cys generally are encoded by preferred reading frames of rearranging D segments, providing a clear design for use of preferred reading frame in antigen receptor D regions. These unusual characteristics shared by NAR and unconventional mammalian Ig are most likely the result of convergent evolution at the molecular level.

Amino Acid Sequence↗
PubMed1998

Members of the Arabidopsis actin gene family are widely dispersed in the genome.

Plant genomes are subjected to a variety of DNA turnover mechanisms that are thought to result in rapid expansion and presumable contraction of gene copy number. The evolutionary history of the 10 actin genes in Arabidopsis thaliana is well characterized and can be traced to the origin of vascular plant genomes. Knowledge about the genomic position of each actin gene may be the key to tracing landmark genomic duplication events that define plant families or genera and facilitate further mutant isolation. All 10 actin genes were mapped by following the segregation of cleaved amplified polymorphisms between two ecotypes and identifying actin gene locations among yeast artificial chromosomes. The Arabidopsis actin genes are widely dispersed on four different chromosomes (1, 2, 3, and 5). Even the members of three closely related and recently duplicated pairs of actin genes are unlinked. Several other cytoskeletal genes (profilins, tubulins) that might have evolved in concert with actins were also mapped, but showed few patterns consistent with that evolutionary history. Thus, the events that gave rise to the actin gene family have been obscured either by the duplication of very small genic fragments or by extensive rearrangement of the genome.

Actins↗
PubMed1998

Detection of deleterious genotypes in multigenerational studies. I. Disruptions in individual Arabidopsis actin genes.

Plant actins are involved in numerous cytoskeletal processes effecting plant development, including cell division plane determination, cell elongation, and cell wall deposition. Arabidopsis thaliana has five ancient subclasses of actin with distinct patterns of spatial and temporal expression. To test their functional roles, we identified insertion mutants in three Arabidopsis actin genes, ACT2, ACT4, and ACT7, representing three subclasses. Adult plants homozygous for the act2-1, act4-1, and act7-1 mutant alleles appear to be robust, morphologically normal, and fully fertile. However, when grown as populations descended from a single heterozygous parent, all three mutant alleles were found at extremely low frequencies relative to the wild-type in the F2 generation. Thus, all three mutant alleles appear to be deleterious. The act2-1 mutant allele was found at normal frequencies in the F1, but at significantly lower frequencies than expected in the F2 and F3 generations. These data suggest that the homozygous act2-1/act2-1 mutant adult plants have a reduced fitness in the 2N sporophytic portion of the life cycle, consistent with the vegetative expression of ACT2. These data are interpreted in light of the extreme conservation of plant actin subclasses and genetic redundancy.

Actins↗
PubMed1997

IgM-mediated opsonization and cytotoxicity in the shark.

Two types of cytotoxic reactions have been observed using cells from the nurse shark: spontaneous cytotoxicity mediated by cells of the macrophage lineage and antibody-dependent killing carried out by a different effector cell population. Previous data showed that removal of phagocytic cells using iron particles abolished macrophage-mediated killing, but not antibody-dependent reactions. The current study used single cell assays and showed that the effector of antibody-driven reactions was the neutrophil. Surprisingly, the mechanism of killing was shown to be phagocytosis mediated by both 7S and 19S immunoglobulin M (IgM). Reactions proceeded with as little as 0.01 microg of purified 19S or 7S IgM and were complete within 4-6 h. In contrast, purified immunoglobulin did not adsorb to macrophages and had no effect on target cell binding or cytotoxicity. Pretreatment of cells with cytochalasin D abolished the phagocytic reaction, but not spontaneous cytotoxicity. These data show that antibody-mediated killing results from opsonization and phagocytosis; the mechanism of macrophage killing is currently unknown. In addition, these data show that the shark neutrophil, not the macrophage lineage, carries a receptor for Fc mu.

Animals↗
PubMed1997

The Arabidopsis ACT11 actin gene is strongly expressed in tissues of the emerging inflorescence, pollen, and developing ovules.

ACT11 represents a unique and ancient actin subclass in the complex Arabidopsis actin gene family. We have isolated and characterized the Arabidopsis ACT11 actin gene and examined its expression. Southern blotting with a 5' gene-specific probe showed that ACT11 was a single-copy gene in the genome. Northern analysis with a 3' gene-specific probe and reverse transcriptase-mediated PCR (RT-PCR) using gene-specific primers detected ACT11 mRNA at low levels in seedling, root, leaf, and silique tissue; at moderate levels in the inflorescence stem and flower; and at very high levels in pollen. The 5' region of the ACT11 gene, including the promoter region, the 5'-untranslated leader, the intron within the leader, and the first 19 actin codons, was fused to a beta-glucuronidase (GUS) reporter gene. The expression of the ACT11/GUS fusion was examined histochemically in numerous independent transgenic Arabidopsis plants. Strong ACT11/GUS activity was detected in rapidly elongating tissues and organs (e.g., etiolated hypocotyls, expanding leaves, stems) and in floral organ primordia. As the floral buds developed into mature flowers, strong GUS activity was gradually restricted to mature pollen and developing ovules. ACT11 appears to be the only Arabidopsis actin gene expressed at significant levels in ovule, embryo, and endosperm. The unique expression patterns in reproductive organs and the sequence divergence of the ACT11 actin gene suggest that the ACT11 isovariant plays distinct and required roles during Arabidopsis development.

Actins↗
PubMed1996

A novel "chimeric" antibody class in cartilaginous fish: IgM may not be the primordial immunoglobulin.

Using a degenerate oligonucleotide primer specific for immunoglobulin (Ig) constant type 1 (C-1 set) domain genes, products were amplified by the reverse transcriptase-polymerase chain reaction from nurse shark spleen cDNA. The deduced protein sequence of one of these clones reveals a novel Ig class in cartilaginous fish. A complete mRNA could encode a mature protein bearing an amino-terminal variable (V) domain, followed by six C-1 set domains, and ending in a carboxy-terminal tail typical of secreted IgM, IgA, and the new antigen receptor (NAR). The two amino-terminal C domains are orthologous to IgX (or IgR), an Ig heavy (H) chain class in the skate, and the last four domains are homologous to the carboxy-terminal four domains of NAR. We designate this "chimeric" Ig class IgNARC for Ig new antigen receptor from cartilaginous fish. Like NAR, but unlike shark IgM, IgNARC is encoded by very few V and C genes which apparently are not closely linked. The number of bands that hybridize with exon-specific probes varies with genomic DNA from individual sharks, suggestive of different numbers of IgNARC genes in different animals. A protein of approximately 95 kDa, which is likely to be the IgNARC H chain, is immunoprecipitated with both light chain-specific monoclonal antibodies and with antisera generated to a peptide comprising the IgNARC carboxy-terminal tail. We conclude that the arsenal of secreted antigen receptors in cartilaginous fish is greater than previously believed. In addition, our data cast doubt on the dogma that IgM is the primordial Ig isotype.

Amino Acid Sequence↗
PubMed1996

Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues.

Arabidopsis has a complex and ancient actin gene family encoding six divergent subclasses of proteins. One subclass is represented by ACT2 and ACT8, which encode nearly identical proteins. These two genes differ significantly in flanking and intron sequences and in silent nucleotide positions within codons. Gene-specific RNA gel blot hybridization and reverse transcriptase-mediated polymerase chain reaction (RT-PCR) assays showed that ACT2 and/or ACT8mRNAs were coordinately and strongly expressed in leaves, roots, stems, flowers, pollen, and siliques. Together they account for greater than 80% of the actin mRNA in most Arabidopsis organs. The 5' flanking regions, including the promoter, the mRNA leader exon, an intron in the mRNA leader, and the first 19 codons, were coupled to a beta-glucuronidase (GUS) reporter gene and transformed into Arabidopsis. The ACT2/GUS construct was expressed strongly in nearly all the vegetative tissues in seedlings, juvenile plants, and mature plants. These activities persisted in older tissues. Little or no expression was observed in seed coats, hypocotyls, gynoecia, or pollen sacs. In contrast, the expression of the ACT8/GUS construct was weaker. It was observed only in a subset of the organs and tissues expressing ACT2/GUS and was not significantly expressed in the flower. ACT2, ACT8, and ACT8/GUS mRNAs were present at moderate to high levels in pollen, and yet neither ACT2/GUS nor ACT8/GUS enzyme expression could be detected in pollen. This suggested a mechanism of translational control affecting ACT2 and ACT8 expression in some tissues. The conservation of protein sequence and overlapping patterns of expression, in spite of significant DNA sequence divergence, suggests that the function and regulation of these two genes have been conserved during the evolution of the Brassicaceae.

Actins↗
PubMed1996

The Arabidopsis thaliana ACT4/ACT12 actin gene subclass is strongly expressed throughout pollen development.

Plants contain complex actin gene families composed of several diverse and ancient subclasses of genes. One Arabidopsis actin gene subclass represented by the ACT4 and ACT12 genes has been isolated and characterized. Both actin genes have typical plant actin gene structures, including three small introns interrupting the coding region and an intron within the mRNA leader. Their encoded proteins differ from each other in only one amino acid, whereas they differ in 3-10% of their amino acids from the other five Arabidopsis actin subclasses. They also share a few small blocks of DNA sequence homology in the 5' flanking region near their TATA boxs, but not in their introns, 3' flanking regions, or degenerate positions within codons. Southern analysis with gene-specific probes from 5' flanking sequences showed that both were single copy genes in the genome. Both RNA gel blot analysis with 3' gene-specific probes and reverse transcriptase-mediated polymerase chain reactions (RT-PCR) with gene-specific primers detected low levels of ACT4 and ACT12 mRNAs in flowers and very high levels in pollen. The RT-PCR detected very low levels of these mRNAs in the vegetative organs. The 5' region from both genes, including the promoter region, TATA box, the sequence for the mRNA leader and its intron, and the first 19 actin codons, was fused to a beta-glucuronidase (GUS) reporter gene. Expression of the GUS fusions were examined histochemically in 40 independent transgenic Arabidopsis plants. Expression of the ACT4/GUS fusion was restricted to young vascular tissues, tapetum, and developing and mature pollen. Similar expression patterns in these tissues and cell types were observed for ACT12/GUS fusion, yet unlike ACT4, ACT12 was also strongly expressed in the root cap and in a ring of pericycle tissues during lateral root initiation and early development. The unique expression patterns of the ACT4/ACT12 actin gene subclass are discussed in light of recent data on the other expressed members of the Arabidopsis actin gene family.

Actins↗
PubMed1996

Structure and evolution of the actin gene family in Arabidopsis thaliana.

Higher plants contain families of actin-encoding genes that are divergent and differentially expressed. Progress in understanding the functions and evolution of plant actins has been hindered by the large size of the actin gene families. In this study, we characterized the structure and evolution of the actin gene family in Arabidopsis thaliana. DNA blot analyses with gene-specific probes suggested that all 10 of the Arabidopsis actin gene family members have been isolated and established that Arabidopsis has a much simpler actin gene family than other plants that have been examined. Phylogenetic analyses suggested that the Arabidopsis gene family contains at least two ancient classes of genes that diverged early in land plant evolution and may have separated vegetative from reproductive actins. Subsequent divergence produced a total of six distinct subclasses of actin, and five showed a distinct pattern of tissue specific expression. The concordance of expression patterns with the phylogenetic structure is discussed. These subclasses appear to be evolving independently, as no evidence of gene conversion was found. The Arabidopsis actin proteins have an unusually large number of nonconservative amino acid substitutions, which mapped to the surface of the actin molecule, and should effect protein-protein interactions.

Actins↗
PubMed1996

The arabidopsis ACT7 actin gene is expressed in rapidly developing tissues and responds to several external stimuli.

ACT7 encodes one of the six distinct and ancient subclasses of actin protein in the complex Arabidopsis actin gene family. We determined the sequence and structure of the Arabidopsis thaliana ACT7 actin gene and investigated its tissue-specific expression and regulation. The ACT7 mRNA levels varied by 128-fold among several different tissues and organs. The highest levels of aCT7 mRNA were found in rapidly expanding vegetative organs, the lowest in pollen. A translational fusion with the 5' end of ACT 7 (1.9 kb) joined to the beta-glucuronidase reporter gene was strongly and preferentially expressed in all young, developing vegetative tissues of transgenic Arabidopsis plants. ACT7 was the only Arabidopsis actin gene strongly expressed in the hypocotyl and seed coat. Although no beta-glucuronidase expression was seen in developing ovules or immature seeds, strong expression was seen in dry seeds and immediately after imbibition in the entire seedling. ACT7 was the only Arabidopsis actin gene to respond strongly to auxin, other hormone treatments, light regime, and wounding, and may be the primary actin gene responding to external stimuli. The ACT7 promoter sequence contains a remarkable number of motifs with sequence similarity to putative phytohormone response elements.

Actins↗