Persistence of immunoglobulin heavy chain/c-myc recombination-positive lymphocyte clones in the blood of human immunodeficiency virus-infected homosexual men.

We studied blood lymphocytes of human immunodeficiency virus (HIV)-seropositive and -negative homosexual men for the presence of T(8;14) translocations that recombine c-myc and immunoglobulin heavy-chain (IgH) mu/IgH alpha switch regions. Clones with T(8;14) translocations were detected in 10.5% (12/114) of the HIV-positive and in 2.0% of the 99 uninfected patients. The majority of recombinations were found at a single time point only. Four patients, however, harbored multiple (up to four) and persistent (up to 9 years) translocation-positive cell clones. No correlation between the presence of these aberrant lymphocytes and a later lymphoma could be established. The exon 1/intron 1 region of the recombined c-myc was investigated for the presence of point mutations and these were found in the nonpersistent clones. Additional alterations detected in these clones included duplications and a deletion in the c-myc gene. The pattern of base substitution indicates that they were introduced after the translocation event.

Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the RLGS spot-bombing method.

We have developed a technique for isolating DNA markers tightly linked to a target region that is based on RLGS, named RLGS spot-bombing (RLGS-SB). RLGS-SB allows us to scan the genome of higher organisms quickly and efficiently to identify loci that are linked to either a target region or gene of interest. The method was initially tested by analyzing a C57BL/6-GusS mouse congenic strain. We identified 33 variant markers out of 10,565 total loci in a 4.2-centimorgan (cM) interval surrounding the Gus locus in 4 days of laboratory work. The validity of RLGS-SB to find DNA markers linked to a target locus was also tested on pooled DNA from segregating backcross progeny by analyzing the spot intensity of already mapped RLGS loci. Finally, we used RLGS-SB to identify DNA markers closely linked to the mouse reeler (rl) locus on chromosome 5 by phenotypic pooling. A total of 31 RLGS loci were identified and mapped to the target region after screening 8856 loci. These 31 loci were mapped within 11.7 cM surrounding rl. The average density of RLGS loci located in the rl region was 0.38 cM. Three loci were closely linked to rl showing a recombination frequency of 0/340, which is < 1 cM from rl. Thus, RLGS-SB provides an efficient and rapid method for the detection and isolation of polymorphic DNA markers linked to a trait or gene of interest.

Pax-6 and lens-specific transcription of the chicken delta 1-crystallin gene.

The abundance of delta-crystallin in the chicken eye lens provides an advantageous marker for tissue-specific gene expression during cellular differentiation. The lens-specific expression of the delta 1-crystallin gene is governed by an enhancer in the third intron, which binds a positive (delta EF2) and negative (delta EF1) factor in its core region. Here we show by DNase I footprinting, electrophoretic mobility-shift assays, and cotransfection experiments with the delta 1-promoter/enhancer fused to the chloramphenicol acetyltransferase reporter gene that the delta 1-crystallin enhancer has two adjacent functional Pax-6 binding sites. We also demonstrate by DNase I footprinting that the delta EF1 site can bind the transcription factor USF, raising the possibility that USF may cooperate with Pax-6 in activation of the chicken delta 1- and alpha A-crystallin genes. These data, coupled with our recent demonstration that Pax-6 activates the alpha A-crystallin gene, suggest that Pax-6 may have been used extensively throughout evolution to recruit and express crystallin genes in the lens.

Isolation of a differentially regulated splicing isoform of human NF-E2.

The transcription factor NF-E2 (nuclear factor erythroid 2), interacting via DNA motifs within regulatory regions of several hematopoietic genes, is thought to mediate the enhancer activity of the globin locus control regions. By screening a human fetal liver cDNA library with probes derived from mouse NF-E2, we have isolated a splicing variant of the NF-E2 gene (fNF-E2) that differs in the 5' untranslated region from the previously reported cDNA (aNF-E2). The fNF-E2 isoform is transcribed from an alternative promoter located in the 3' end of the first intron and joined by alternative splicing to the second and third exons, which are shared by both RNA isoforms. Although the two forms produce the same protein, they are expressed in different ratios during development. fNF-E2 is more abundant in the fetal liver and less abundant in the adult bone marrow compared to the previously described form. Their distribution apparently follows the differential expression of fetal and adult hemoglobins.

Modulation of 5' splice site choice in pre-messenger RNA by two distinct steps.

Ser/Arg-rich proteins (SR proteins) are essential splicing factors that commit pre-messenger RNAs to splicing and also modulate 5' splice site choice in the presence or absence of functional U1 small nuclear ribonucleoproteins (snRNPs). Here, we perturbed the U1 snRNP in HeLa cell nuclear extract by detaching the U1-specific A protein using a 2'-O-methyl oligonucleotide (L2) complementary to its binding site in U1 RNA. In this extract, the standard adenovirus substrate is spliced normally, but excess amounts of SR proteins do not exclusively switch splicing from the normal 5' splice site to a proximal site (site 125 within the adenovirus intron), suggesting that modulation of 5' splice site choice exerted by SR proteins requires integrity of the U1 snRNP. The observation that splicing does not necessarily follow U1 binding indicates that interactions between the U1 snRNP and components assembled on the 3' splice site via SR proteins may also be critical for 5' splice site selection. Accordingly, we found that SR proteins promote the binding of the U2 snRNP to the branch site and stabilize the complex formed on a 3'-half substrate in the presence or absence of functional U1 snRNPs. A novel U2/U6/3'-half substrate crosslink was also detected and promoted by SR proteins. Our results suggest that SR proteins in collaboration with the U1 snRNP function in two distinct steps to modulate 5' splice site selection.

Fate of a redundant gamma-globin gene in the atelid clade of New World monkeys: implications concerning fetal globin gene expression.

Conclusive evidence was provided that gamma 1, the upstream of the two linked simian gamma-globin loci (5'-gamma 1-gamma 2-3'), is a pseudogene in a major group of New World monkeys. Sequence analysis of PCR-amplified genomic fragments of predicted sizes revealed that all extant genera of the platyrrhine family Atelidae [Lagothrix (woolly monkeys), Brachyteles (woolly spider monkeys), Ateles (spider monkeys), and Alouatta (howler monkeys)] share a large deletion that removed most of exon 2, all of intron 2 and exon 3, and much of the 3' flanking sequence of gamma 1. The fact that two functional gamma-globin genes were not present in early ancestors of the Atelidae (and that gamma 1 was the dispensible gene) suggests that for much or even all of their evolution, platyrrhines have had gamma 2 as the primary fetally expressed gamma-globin gene, in contrast to catarrhines (e.g., humans and chimpanzees) that have gamma 1 as the primary fetally expressed gamma-globin gene. Results from promoter sequences further suggest that all three platyrrhine families (Atelidae, Cebidae, and Pitheciidae) have gamma 2 rather than gamma 1 as their primary fetally expressed gamma-globin gene. The implications of this suggestion were explored in terms of how gene redundancy, regulatory mutations, and distance of each gamma-globin gene from the locus control region were possibly involved in the acquisition and maintenance of fetal, rather than embryonic, expression.

RNA-Seq expression profile of genes related to neurodegenerative disorders affecting the human retina

Human neurodegenerative diseases, such as Parkinson’s disease (PD) and the neuromuscular disorders called dystroglycanopathies (DGPs), cause retinal impairments. We have used RNA-Seq technology to catalog all known genes linked to PD and DGPs expressed in the human retina and quantitate their mRNA levels in terms of FPKM. We have also characterized their expression profiles in the retina by determining their exonic, intronic and exon-intron junction expression levels, as well as the alternative splicing pattern of particular genes. We believe these data could pave the way toward understanding the molecular bases of sight deficiencies associated with neurodegenerative disorders.

The Relationship Between Cadwaladerite Al(OH)2Cl∙4H2O and Lesukite Al2(OH)5Cl∙2H2O as Investigated by SEM, PXRD, FTIR and Raman Spectroscopy

Cadwaladerite (Al(OH)2Cl∙4H2O) collected from Cerro Pintados, Chile described by Gordon in 1941 is designated as “doubtful” by the IMA. Material collected from the same locality in 2015 resembling the description of cadwaladerite gave a powder XRD pattern similar to lesukite (Al2(OH)5Cl∙2H2O). However, Gordon provided no X-ray data for his material from Cerro Pintados. In order to determine whether cadwaladerite and lesukite are the same mineral species, measurements were made on a suite of samples from various localities. A portion of the material collected by Gordon in 1941 was also obtained from the Mineralogical Museum of Harvard University. Type material of lesukite from a fumarolic environment at the Tolbachik Fissure in Kamchatka, Russia was obtained as well as lesukite from the Maria Mine, Chile (Arica Province) and a previously undescribed locality for lesukite (Barranaca del Sulfato, Mejillones Peninsula, Antofagasta Province). All samples are yellow to yellow-orange in colour and all exhibit small cubic crystals (up to 50µm), even Gordon’s cadwaladerite which was thought to be amorphous. The Chilean samples are all associated with halite and sometimes with anhydrite. These five samples were studied by SEM, FTIR, powder XRD, and Raman spectroscopy. A ratio of Al:Cl less than or equal to 1.3:1 was observed for all the samples, including measurements made on lesukite from the Russian locality Vergasova et al. studied in 1997, and determined to have a 2:1 ratio. SEM-EDS analyses also show all samples to have minor iron substitution, as well as copper substitution in two samples. FTIR spectra are very similar for all samples. Raman spectroscopy done on both samples collected in Cerro Pintados and the Russian lesukite gave similar spectra. Powder XRD analyses on all samples showed spectra identified to be lesukite, including Gordon’s cadwaladerite. Crystal cell parameters calculated from powder XRD ranged from 19.778Å to 19.878Å. Results using modern instrumental techniques confirm Gordon’s cadwaladerite, collected in 1939 and described in 1941, and lesukite are the same mineral species.

Genetic association of CD247 (CD3ζ) with SLE in a large-scale multiethnic study

A classic T-cell phenotype in systemic lupus erythematosus (SLE) is the downregulation and replacement of the CD3ζ chain that alters T-cell receptor signaling. However, genetic associations with SLE in the human CD247 locus that encodes CD3ζ are not well established and require replication in independent cohorts. Our aim was therefore to examine, localize and validate CD247-SLE association in a large multiethnic population. We typed 44 contiguous CD247 single-nucleotide polymorphisms (SNPs) in 8922 SLE patients and 8077 controls from four ethnically distinct populations. The strongest associations were found in the Asian population (11 SNPs in intron 1, 4.99 × 10(-4) < P < 4.15 × 10(-2)), where we further identified a five-marker haplotype (rs12141731-rs2949655-rs16859085-rs12144621-rs858554; G-G-A-G-A; P(hap) = 2.12 × 10(-5)) that exceeded the most associated single SNP rs858554 (minor allele frequency in controls = 13%; P = 4.99 × 10(-4), odds ratio = 1.32) in significance. Imputation and subsequent association analysis showed evidence of association (P < 0.05) at 27 additional SNPs within intron 1. Cross-ethnic meta-analysis, assuming an additive genetic model adjusted for population proportions, showed five SNPs with significant P-values (1.40 × 10(-3) < P< 3.97 × 10(-2)), with one (rs704848) remaining significant after Bonferroni correction (P(meta) = 2.66 × 10(-2)). Our study independently confirms and extends the association of SLE with CD247, which is shared by various autoimmune disorders and supports a common T-cell-mediated mechanism.

Next-generation sequencing of iron-metabolism related genes in Portuguese patients with iron overload: novel pathogenic genetic variants

Objective: In Southern European countries up to one-third of the patients with hereditary hemochromatosis (HH) do not present the common HFE risk genotype. In order to investigate the molecular basis of these cases we have designed a gene panel for rapid and simultaneous analysis of 6 HH-related genes (HFE, TFR2, HJV, HAMP, SLC40A1 and FTL) by next-generation sequencing (NGS). Materials and Methods: Eighty-eight iron overload Portuguese patients, negative for the common HFE mutations, were analysed. A TruSeq Custom Amplicon kit (TSCA, by Illumina) was designed in order to generate 97 amplicons covering exons, intron/exon junctions and UTRs of the mentioned genes with a cumulative target sequence of 12115bp. Amplicons were sequenced in the MiSeq instrument (IIlumina) using 250bp paired-end reads. Sequences were aligned against human genome reference hg19 using alignment and variant caller algorithms in the MiSeq reporter software. Novel variants were validated by Sanger sequencing and their pathogenic significance were assessed by in silico studies. Results: We found a total of 55 different genetic variants. These include novel pathogenic missense and splicing variants (in HFE and TFR2), a very rare variant in IRE of FTL, a variant that originates a novel translation initiation codon in the HAMP gene, among others. Conclusion: The merging of TSCA methodology and NGS technology appears to be an appropriate tool for simultaneous and fast analysis of HH-related genes in a large number of samples. However, establishing the clinical relevance of NGS-detected variants for HH development remains a hard-working task, requiring further functional studies.

Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis

Long distance transport of amino acids is mediated by several families of differentially expressed amino acid transporters. The two genes AAP1 and AAP2 encode broad specificity H+-amino acid co-transporters and are expressed to high levels in siliques of Arabidopsis, indicating a potential role in supplying the seeds with organic nitrogen. The expression of both genes is developmentally controlled and is strongly induced in siliques at heart stage of embryogenesis, shortly before induction of storage protein genes. Histochemical analysis of transgenic plants expressing promoter-GUS fusions shows that the genes have non-overlapping expression patterns in siliques. AAP1 is expressed in the endosperm and the cotyledons whereas AAP2 is expressed in the vascular strands of siliques and in funiculi. The endosperm expression of AAP1 during early stages of seed development indicates that the endosperm serves as a transient storage tissue for organic nitrogen. Amino acids are transported in both xylem and phloem but during seed filling are imported only via the phloem. AAP2, which is expressed in the phloem of stems and in the veins supplying seeds, may function in uptake of amino acids assimilated in the green silique tissue, in the retrieval of amino acids leaking passively out of the phloem and in xylem-to-phloem transfer along the path. The promoters provide excellent tools to study developmental, hormonal and metabolic control of nitrogen nutrition during development and may help to manipulate the timing and composition of amino acid import into seeds.

Lethal chondrodysplasia in a family of Holstein cattle is associated with a de novo splice site variant of COL2A1

Background Lethal chondrodysplasia (bulldog syndrome) is a well-known congenital syndrome in cattle and occurs sporadically in many breeds. In 2015, it was noticed that about 12 % of the offspring of the phenotypically normal Danish Holstein sire VH Cadiz Captivo showed chondrodysplasia resembling previously reported bulldog calves. Pedigree analysis of affected calves did not display obvious inbreeding to a common ancestor, suggesting the causative allele was not a rare recessive. The normal phenotype of the sire suggested a dominant inheritance with incomplete penetrance or a mosaic mutation. Results Three malformed calves were examined by necropsy, histopathology, radiology, and computed tomography scanning. These calves were morphologically similar and displayed severe disproportionate dwarfism and reduced body weight. The syndrome was characterized by shortening and compression of the body due to reduced length of the spine and the long bones of the limbs. The vicerocranium had severe dysplasia and palatoschisis. The bones had small irregular diaphyses and enlarged epiphyses consisting only of chondroid tissue. The sire and a total of four affected half-sib offspring and their dams were genotyped with the BovineHD SNP array to map the defect in the genome. Significant genetic linkage was obtained for several regions of the bovine genome including chromosome 5 where whole genome sequencing of an affected calf revealed a COL2A1 point mutation (g.32473300 G > A). This private sequence variant was predicted to affect splicing as it altered the conserved splice donor sequence GT at the 5’-end of COL2A1 intron 36, which was changed to AT. All five available cases carried the mutant allele in heterozygous state and all five dams were homozygous wild type. The sire VH Cadiz Captivo was shown to be a gonadal and somatic mosaic as assessed by the presence of the mutant allele at levels of about 5 % in peripheral blood and 15 % in semen. Conclusions The phenotypic and genetic findings are comparable to a previously reported COL2A1 missense mutation underlying lethal chondrodysplasia in the offspring of a mosaic French Holstein sire (Igale Masc). The identified independent spontaneous splice site variant in COL2A1 most likely caused chondrodysplasia and must have occurred during the early foetal development of the sire. This study provides a first example of a dominant COL2A1 splice site variant as candidate causal mutation of a severe lethal chondrodysplasia phenotype. Germline mosaicism is a relatively frequent mechanism in the origin of genetic disorders and explains the prevalence of a certain fraction of affected offspring. Paternal dominant de novo mutations are a risk in cattle breeding, especially because the ratio of defective offspring may be very high and be associated with significant animal welfare problems.

1964 Ice Hockey Team

University of Michigan 150-pound football team, 1948

Top Row: Dick Bodycomb, Gus Eaton, Bud Marshall, Tony Cybulski, Herb Taggart, Bob Kash, Burl Selden (ass't coach), Cliff Keen (coach).

Third Row: Gene Kiddon, Gordon Smith, Vaughn Lowther, Jim Coleman, Larry Shaw, James Johnson, Frank Whitehouse, Gene Freed, Everet Knapp, George Sipp, Stan Emerling.

Second Row: Bill Clark, James Richter, Jack Keller, Dave Bradbury Fred Koehn, Hyman Berman, Bill Parshall, Bill Connoly, M. Richard Fleishman, Frank Murphy, Jim Briskin, Leon Hinz.

Bottom Row: John Wilcox, Prentice Ryan, Ed Morey, Jim Costa, Barry Breakey, Jere Ogle, Jim Armelagos, Don O'Connell, Jerry Burns, John Picard, James Sakai, Ted Karmazin.

UM Men's Swimming Team, 1963UM Swimming

Front Row, Left to Right: Paul A. Attar, Michael G. Reissing, Steven D. Thrasher, captain John H. Dumont, Stanley C. (Pete) Cox, Richard F. Nelson.

Second Row: Warren G. Uhler, Roy D. Burry, John Candler, Thomas Burns, Andrew Paterson, Patrick Faul, Jon W. Lundin.,

Back Row: swimming coach Gus Stager, Stephen Selander, Frederick R. Damm, Dennis J. Fitzgerald, Richard Abineri, Jerry Traver, diving coach Dick Kimball.