A map of the human immunoglobulin VH locus completed by analysis of the telomeric region of chromosome 14q.

Analysis of the telomeric region of chromosome 14q has enabled us to complete a map of the immunoglobulin VH locus which accounts for almost all VH segments known to rearrange in B-lymphocytes. The human germline VH repertoire consists of approximately 50 functional VH segments--the exact number depending on the haplotype--spanning 1,100 kilobases upstream of the JH segments. A yeast artificial chromosome used to map these segments was isolated by its ability to provide telomere activity in yeast, suggesting that the VH locus may be located within a few kilobases of the 14q telomere. The limited structural diversity encoded by the functional VH segments demonstrates the importance of combinatorial diversity produced by VDJ joining and the association of heavy and light chains in producing the human antibody repertoire.

Human subtelomeric copy number variations.

Copy number variation is a defining characteristic of human subtelomeres. Human subtelomeric segmental duplication regions ('Subtelomeric Repeats') comprise about 25% of the most distal 500 kb and 80% of the most distal 100 kb in human DNA. Huge allelic disparities seen in subtelomeric DNA sequence content and organization are postulated to have an impact on the dosage of transcripts embedded within the duplicated sequences, on the transcription of genes in adjacent single copy DNA regions, and on the chromatin structures mediating telomere functions including chromosome stability. In addition to the complex duplicon substructure and huge allelic variations in extended subtelomere regions, both copy number variation and alternative sequence organizations for DNA characterize the sequences immediately adjacent to terminal (TTAGGG)n tracts ('subterminal DNA'). The structural variation in subterminal DNA is likely to have important consequences for expression of subterminal transcripts such as a newly-discovered gene family encoding actin-interacting proteins and a non-coding telomeric repeat containing RNA (TERRA) transcript family critical for telomere integrity. Major immediate challenges include discovering the full extent and nature of subtelomeric structural and copy number variation in humans, and developing methods for tracking individual allelic variants in the context of total genomic DNA.

Metaphase and interphase cytogenetics with Alu-PCR-amplified yeast artificial chromosome clones containing the BCR gene and the protooncogenes c-raf-1, c-fms, and c-erbB-2.

A human yeast artificial chromosome (YAC) library was screened by polymerase chain reaction with oligonucleotide primers defined for DNA sequences of the BCR gene and the protooncogenes c-raf-1, c-fms, and c-erbB-2. Alu-PCR-generated human DNA sequences were obtained from the respective YAC clones and used for fluorescence in situ hybridization experiments under suppression conditions. After chromosomal in situ suppression hybridization to GTG-banded human prometaphase chromosomes, seven of nine initially isolated YAC clones yielded strong signals exclusively in the chromosome bands containing the respective genes. Two clones yielded additional signals on other chromosomes and were excluded from further tests. The band-specific YACs were successfully applied to visualize specific structural chromosome aberrations in peripheral blood cells from patients with myelodysplasia exhibiting del(5)(q13q34), chronic myeloid leukemia and acute lymphocytic leukemia with t(9;22)(q34;q11), acute promyelocytic leukemia (M3) with t(15;17)(q22;q21), and in a cell line established from a proband with the constitutional translocation t(3;8)(p14.2;q24). In addition to the analysis of metaphase spreads, we demonstrate the particular usefulness of these YAC clones in combination with whole chromosome painting to analyze specific chromosome aberrations directly in the interphase nucleus.

Purification and characterization of an iron stress-induced chlorophyll-protein from the cyanobacterium Anacystis nidulans R2.

An Anacystis nidulans R2 chlorophyll-protein associated with Photosystem II in iron-stressed cells (Pakrasi, H.B., Riethmann, H.C. and Sherman, L.A. (1985) Proc. Natl. Acad. Sci. USA 82, 6903-6907) has been biochemically purified and characterized. Anion exchange chromatography of dodecyl-beta-D-maltoside-solubilized membranes from iron-deficient cells was used to recover this chlorophyll-protein (termed CPVI-4) in high yield and in a relatively native state. CPVI-4 has a room temperature absorption maximum at 671 nm, a 77 K chlorophyll fluorescence peak at 681 nm, and contains polypeptides of 36, 34 and 12 kDa. The 36 and 34 kDa polypeptides are associated with chlorophyll on mildly denaturing acrylamide gels of purified CPVI-4, although only the 34 kDa protein is immunoreactive with antisera elicited against the gel-purified chlorophyll-protein. Immunoblotting experiments with dodecyl-beta-D-maltoside-solubilized membrane fractions and purified CPVI-4 indicate that CPVI-4 does not contain previously identified Photosystem II core proteins. CPVI-4 likely functions as a light-harvesting antenna complex in iron-starved cells (where phycobilisomes are absent or diminished) and, in addition, may contribute chlorophyll to the reaction center complexes during their assembly in the early stages of recovery from iron stress.

Organization of the thylakoid membrane from the heterotrophic cyanobacterium, Aphanocapsa 6714.

The polypeptide composition of thylakoid membrane fractions from the heterotrophic cyanobacterium Aphanocapsa 6714 was examined by electrophoretic and immunoblotting procedures. We have identified thylakoid cytochromes f, b6, c-550 and c-553 by tetramethylbenzidine staining of lithium dodecyl sulfate polyacrylamide gels; we also have identified the Rieske Fe-S center protein and subunit 4 of the cytochrome b6/f complex. We have characterized phycobilisomes and active core preparations of PS I and PS II. PS I is comprised of five polypeptides (62 kDa, 14.5 kDa, 10 kDa, and two proteins of less than 10 kDa), and our PS II preparation is highly enriched for three chlorophyll-binding proteins of 48, 45 and 36 kDa. Furthermore, we have resolved the chlorophyll-binding complexes on non-denaturing gels and have determined the polypeptide composition of each chlorophyll-containing band. Three bands are associated with PS I (I, IIa and IIb) and three bands are PS II components (III', IIIa and IIIb) as judged by low-temperature fluorescence emission spectra. Band III' contains a 64 kDa antenna polypeptide, IIIa contains the 48 kDa and 45 kDa polypeptides, and IIIb is comprised solely of a 36 kDa protein. The IIIb apoprotein represents a novel PS II component; its possible role in photochemistry is discussed.

Phycobilisome-associated glycoproteins in the cyanobacterium Anacystis nidulans R 2.

Four phycobilisome-associated proteins were found to be specifically reactive with the lectin concanavalin A after subunits of isolated Anacystis nidulans R 2 phycobilisomes were separated on polyacrylamide gels and transferred onto nitrocellulose. The concanavalin A-reactive phycobilisome components have proposed functions related to the orientation, assembly, and membrane attachment of the phycobilisome. Chemical analysis of the total isolated phycobilisome material indicated the presence of glucose (approx. 1.5% by wt) and N-acetylgalactosamine (0.15% by wt), consistent with our proposal that the concanavalin A-reactive polypeptides contain covalently linked, glucose-containing polysaccharides.

Human subtelomere structure and variation.

Work towards completion of the human reference genome sequence has revealed a great deal of complexity and plasticity in human subtelomeric regions. The highly variable subtelomeric repeat regions are filled with recently shuffled genomic segments, many of which contain sequences matching transcripts and transcript fragments; the rapid duplication and combinatorial evolution of these regions has generated an extremely diverse set of subtelomeric alleles in the human species, the complexity and potential significance of which is only beginning to be understood. This review summarizes recent progress in analyzing human subtelomeric sequence assemblies and large-scale variation in human subtelomere regions.

Immunological Characterization of Iron-Regulated Membrane Proteins in the Cyanobacterium Anacystis nidulans R2.

Antibodies cross-reactive with specific membrane proteins were used to investigate membrane development in Anacystis nidulans R2 during recovery from iron stress. Polyclonal antibodies prepared using the iron-regulated chlorophyll (Chl)-protein CPVI-4 (HB Pakrasi, HC Riethman, LA Sherman 1985 Proc Natl Acad Sci USA 82: 6903-6907) as antigen were characterized and used to identify three iron stress-induced polypeptides of 36, 35, and 34 kilodaltons on immunoblots of polyacrylamide gels. The 34 kilodalton protein was shown to be a component of the Chlbinding CPVI-4 complex. The 36 kilodalton protein is an unrelated, intrinsic membrane protein tightly regulated by iron (designated IrpA), whereas the 35 kilodalton immunoreactive component is an extremely abundant glycoprotein (GP35). An analysis of photosystem II (PSII)-associated Chl-proteins during recovery from iron stress demonstrates that CPVI-4 is associated with most of the Chl present in iron-starved cells, whereas the PSII core polypeptides are present in very low levels; upon recovery, CPVI-4 diminishes in abundance as the relative levels of the other PSII proteins increase. The abundance of CPVI-4 in iron-stressed cells and the distribution of Chl among individual Chl-proteins during recovery suggest a possible role for CPVI-4 in the direction of membrane assembly during recovery from iron stress.

Direct DNA hybridization screening of primary yeast transformants in the construction of targeted yeast artificial chromosome (YAC) libraries.

A simple and inexpensive method for direct, large-scale DNA hybridization screening of primary yeast colonies following introduction of foreign DNA into Saccharomyces cerevisiae by spheroplast transformation is presented. An optimally thin layer of standard regeneration agar containing the transformed spheroplasts is spread on selective plates using carefully controlled temperature conditions; the colonies regenerate initially within the thin layer of hardened regeneration agar, but as they grow, greater than 90% of them break the surface and become accessible to direct lifting and screening methods. The technique avoids the manual or mechanized picking of transformants from within the regeneration top agar, does not require specialized (and often expensive) regeneration matrices such as alginate or low-melting-point agarose, and yields transformation efficiencies and screening results identical to those obtained using the standard spheroplast transformation protocol. We demonstrate the utility of this method for the construction of a targeted human-YAC library from a hamster hybrid cell line that contains chromosome 13 as its only human DNA.

Painting of human chromosomes with probes generated from hybrid cell lines by PCR with Alu and L1 primers.

Specific amplification of human sequences of up to several kb length has recently been accomplished in man-hamster and man-mouse somatic hybrid cell DNA by IRS-PCR (interspersed repetitive sequence - polymerase chain reaction). This approach is based on oligonucleotide primers that anneal specifically to human Alu- or L1-sequences and allows the amplification of any human sequences located between adequately spaced, inverted Alu- or L1-blocks. Here, we demonstrate that probe pools generated from two somatic hybrid cell lines by Alu- and L1-PCR can be used for chromosome painting in normal human lymphocyte metaphase spreads by chromosomal in situ suppression (CISS-) hybridization. The painted chromosomes and chromosome subregions directly represent the content of normal and deleted human chromosomes in the two somatic hybrid cell lines. The combination of IRS-PCR and CISS-hybridization will facilitate and improve the cytogenetic analysis of somatic hybrid cell panels, in particular, in cases where structurally aberrant human chromosomes or human chromosome segments involved in interspecies translocations cannot be unequivocally identified by classical banding techniques. Moreover, this new approach will help to generate probe pools for the specific delineation of human chromosome subregions for use in cytogenetic diagnostics and research without the necessity of cloning.

Characterization of phycobilisome glycoproteins in the cyanobacterium Anacystis nidulans R2.

Concanavalin A-reactive linker and anchor subunits of phycobilisomes from Anacystis nidulans R2 (H. C. Riethman, T. P. Mawhinney, and L. A. Sherman, FEBS Lett. 215:209-214, 1987) were purified electrophoretically and analyzed for carbohydrate composition and quantity. Different quantities of glucose and N-acetylgalactosamine were found on the concanavalin A-reactive subunits analyzed. Proteolytic analysis of the purified subunits suggested that small regions of the 33- and 27-kilodalton linker polypeptides previously shown to be important for in vitro phycobilisome assembly contained the concanavalin A-reactive carbohydrates present on these subunits. The linker and anchor subunits from the morphologically different phycobilisome of Synechocystis sp. strain PCC6714 were also shown to be concanavalin A reactive. Membranes from iron-starved Anacystis nidulans, which lack assembled phycobilisomes and are associated with glycogen deposits, were shown to be depleted of linker and anchor proteins and to accumulate very large quantities of a concanavalin A-reactive, extrinsic membrane glycoprotein. We suggest that this iron stress-induced glycoprotein is associated with the glycogen deposits on the thylakoid surface and that the glycosylation of phycobilisome linker and anchor subunits is involved in the physiological regulation of phycobilisome assembly and degradation.

Organization of pigment proteins in the photosystem II complex of the cyanobacterium Anacystis nidulans R2.

Two chlorophyll-protein complexes associated with photosystem II (PSII) of the cyanobacterium Anacystis nidulans R2 have been detected. The larger of the two complexes, CPVI-1, contained a 71-kDa and a 42-kDa protein. The 71-kDa protein was determined to be the anchor protein of the phycobilisomes (the light-harvesting complex of A. nidulans PSII), since it was recognized by an antibody raised against a similar protein from another cyanobacterium. The second complex, CPVI-4, contained a previously unobserved 36-kDa chlorophyll-binding protein. Additionally, two other PSII chlorophyll-protein bands were characterized. CPVI-2 contained a 52-kDa band that was recognized by an antibody raised against the presumptive PSII reaction center protein of Chlamydomonas reinhardtii. It gave rise to a fluorescence emission peak (77K) at 695 nm, indicating that this chlorophyll-protein complex may harbor the reaction center of PSII. Finally, CPVI-3 was found to have a 45-kDa protein and to be immunologically related to the presumptive immediate-antenna protein of the C. reinhardtii PSII.

Isolation and Characterization of a Carotenoid-Associated Thylakoid Protein from the Cyanobacterium Anacystis nidulans R2.

A carotenoid-associated membrane protein was isolated from Anacystis nidulans R2 thylakoids. Sodium pyrophosphate and sodium bromide washed thylakoids were solubilized with the nonionic detergents dodecyl-beta-D-maltoside and octyl-beta-D-glucopyranoside, and these detergent extracts were fractionated on a sucrose density gradient. A yellow fraction from the sucrose gradient was further purified by anion-exchange and organomercuric-affinity column chromatography to yield a fraction virtually free of chlorophyll and highly enriched in both carotenoids and a 42 kilodalton polypeptide. Evidence presented in this paper suggests that the carotenoid-containing 42 kilodalton protein is thylakoid associated rather than cytoplasmic membrane associated. The purified 42 kilodalton polypeptide was used to raise polyclonal antibodies in rabbits. Immuno-chemical detection of the 42 kilodalton polypeptide on Western blots demonstrated an increased accumulation of this polypeptide in cells grown under high-light conditions relative to cells grown under low light.

Triton X-114 phase fractionation of an integral membrane surface protein mediating monoclonal antibody killing of Mycoplasma hyorhinis.

A previously defined immunoglobulin M(kappa) monoclonal antibody reacting with a surface epitope of Mycoplasma hyorhinis is shown in this report to mediate specific, complement-dependent mycoplasmacidal activity. Immunoblot analysis of mycoplasma components and their tryptic cleavage products showed that the epitope recognized was present on a protein with an apparent molecular weight of 23,000 (p23) and on a limit tryptic fragment of this protein with an apparent molecular weight of 18,000 (p18). Both p23 and p18 are shown by Triton X-114 phase fractionation to partition efficiently into the hydrophobic detergent phase. Other antigens bearing epitopes not expressed at the cell surface were present among the numerous hydrophilic proteins found in the aqueous phase. The external orientation and membrane association of the p23 antigen were further established by demonstrating that trypsin treatment of intact mycoplasmas generated the antigenic p18 fragment, which remained tightly associated with the organism. These results localize an epitope responsible for antibody-mediated mycoplasma killing onto a specific, surface-exposed region of an integral membrane protein of this organism. Since the monoclonal antibody used in this study does not bind to the surface of all strains of M. hyorhinis, the epitope identified also defines a structural marker of antigenic surface variation within this species, a feature previously observed during serological classification of the organism. Analysis of the antigenic and structural features of the p23 surface antigen may therefore be useful in establishing mechanisms of surface antigen variation among integral membrane proteins of mycoplasmas that could dictate important antigenic characteristics recognized during chronic disease caused by these agents.

Detection and characterization of chimeric yeast artificial-chromosome clones.

Methods for the construction of yeast artificial-chromosome (YAC) clones have been designed to isolate single, large (100-1000 kb) segments of chromosomal DNA. It is apparent from early experience with this cloning system that the major artifact in YAC clones involves the formation of YACs that contain two or more unrelated pieces of DNA. Such "chimeric" YACs are not easily recognized, particularly in libraries constructed from the total DNA of an organism. In some libraries, they have been found to constitute a major fraction of the clones. Here we discuss some of our experiences with chimeric YACs, with particular emphasis on the approaches that we have employed to detect such aberrant clones. In addition, we describe the detailed characterization of one chimeric YAC isolated from a library prepared from total human DNA. The organization of this clone indicates that it formed by in vivo recombination, presumably in yeast, between two Alu sequences located on unrelated segments of human DNA.

Regulation of cyanobacterial pigment-protein composition and organization by environmental factors.

The coordinate expression of stress-specific genes is a common response of all organisms to altered environmental conditions. In cyanobacteria, the physiological consequences of stress are often reflected in both the ultrastructure of the cell and in photosynthesis-related properties. This review will focus on the alterations in cyanobacterial pigment-protein organization which occur under different growth conditions, and how several molecular genetic aproaches are being used in this laboratory to investigate the regulatory mechanisms underlying these alterations. We will discuss in detail the response to iron starvation, and present a testable hypothesis for the mechanism of thylakoid reorganization mediated by this response.

Cloning human telomeric DNA fragments into Saccharomyces cerevisiae using a yeast-artificial-chromosome vector.

Telomeric fragments of human DNA ranging in size from 50 to 250 kilobases were cloned into Saccharomyces cerevisiae using a yeast-artificial-chromosome (YAC) vector. Six human-telomeric YAC (HTY) strains were selected by virtue of the specific hybridization of their DNA with the human telomeric terminal-repeat sequence (TTAGGG)n, and the telomeric localization of this sequence within each YAC was demonstrated by its sensitivity to nuclease BAL-31. In situ hybridization of DNA from three of these HTY strains with human metaphase chromosomes yielded discrete patterns of hybridization signals at the telomeres of a limited number of human chromosomes, different for each clone. DNA from selected cosmid subclones of one of the HTY strains was used to localize the origin of the cloned telomeric DNA by in situ hybridization to the tip of the long arm of chromosome 7.

Characterization of polymorphic loci on a telomeric fragment of DNA from the long arm of human chromosome 7.

The 240-kb yeast artificial chromosome (YAC) HTY146 (D7S427) containing the telomere from the q arm of human chromosome 7 was subcloned into the cosmid vector sCOS-1. Cosmid subclones were screened for DNA polymorphisms by Southern blot analysis of restriction digests of DNA from random individuals. Four distinct polymorphisms were characterized. These markers provide a resource for defining the end of the genetic map for the long arm of human chromosome 7.