Distribution and intensity of constraint in mammalian genomic sequence

Comparisons of orthologous genomic DNA sequences can be used to characterize regions that have been subject to purifying selection and are enriched for functional elements. We here present the results of such an analysis on an alignment of sequences from 29 mammalian species. The alignment captures approximately 3.9 neutral substitutions per site and spans approximately 1.9 Mbp of the human genome. We identify constrained elements from 3 bp to over 1 kbp in length, covering approximately 5.5% of the human locus. Our estimate for the total amount of nonexonic constraint experienced by this locus is roughly twice that for exonic constraint. Constrained elements tend to cluster, and we identify large constrained regions that correspond well with known functional elements. While constraint density inversely correlates with mobile element density, we also show the presence of unambiguously constrained elements overlapping mammalian ancestral repeats. In addition, we describe a number of elements in this region that have undergone intense purifying selection throughout mammalian evolution, and we show that these important elements are more numerous than previously thought. These results were obtained with Genomic Evolutionary Rate Profiling (GERP), a statistically rigorous and biologically transparent framework for constrained element identification. GERP identifies regions at high resolution that exhibit nucleotide substitution deficits, and measures these deficits as "rejected substitutions". Rejected substitutions reflect the intensity of past purifying selection and are used to rank and characterize constrained elements. We anticipate that GERP and the types of analyses it facilitates will provide further insights and improved annotation for the human genome as mammalian genome sequence data become richer.     

Cloning and sequence analysis of the glucoamylase gene of Neurospora crassa

A 1.0-kb DNA fragment, corresponding to an internal region of the Neurospora crassa glucoamylase gene, gla-1, was generated from genomic DNA by the polymerase chain reaction, using oligonucleotide primers which had been deduced from the known N-terminal amino-acid sequence or from consensus regions within the aligned amino-acid sequences of other fungal glucoamylases. The fragment was used to screen an N. crassa genomic DNA library. One clone contained the gene together with flanking regions and its sequence was determined. The gene was found to code for a preproprotein of 626 amino acids, 35 of which constitute a signal and propeptide region. The protein and the gene are compared with corresponding sequences in other fungi.     

Desensitization in vitro—The specific inhibition, by antigen, of the passive transfer of delayed hypersensitivity by peritoneal exudate cells

A preliminary experiment showed that the injection of bovine γ-globulin into guinea-pigs with delayed hypersensitivity to bovine γ-globulin reduced the 24-hour skin reactions to bovine γ-globulin and (to a lesser extent) PPD. The peritoneal exudate cells from the desensitized donors had a reduced ability to transfer delayed hypersensitivity to bovine γ-globulin but a normal ability to transfer delayed hypersensitivity to PPD.

Likewise, it was possible to diminish the passive transfer of delayed hypersensitivity to bovine γ-globulin by peritoneal exudate cells, by exposure of the cells to bovine γ-globulin in vitro. The recipients were tested immediately after cell transfer. This in vitro desensitization was specific, in that the transfer of delayed hypersensitivity to PPD was unaffected.

Exposure of cells in vitro to hypotonic conditions and antibody to guinea-pig γ-globulin did not prevent the passive transfer of delayed hypersensitivity.

     

Studies of human myeloid antigens using monoclonal antibodies and variant lines from the promyeloid cell line HL60.

Monoclonal antibodies were produced using mice immunized with the human promyeloid cell line HL60. Two antibodies are described which identify antigens selectively expressed by myeloid cells. Studies using normal bone marrow and myeloid leukaemia cells demonstrated that one of these antibodies (AGF4.48) identifies an antigen expressed throughout the promyeloid to neutrophil stages of maturation. In contrast, the second antibody (AGF4.36) identifies an antigen expressed at the promyeloid to metamyeloid stages and is absent from most blood neutrophils. The HL60 line can be induced to differentiate into neutrophils by 1 . 25% dimethylsulphoxide (DMSO) (Collins et al., 1978). Variant lines from HL60, unresponsive to 1 . 25% DMSO, lack the 'transient' myeloid antigen (AGF4.36) and show a reduced expression of the myeloid antigen (AGF4.48). The variant lines can be induced to mature using higher DMSO concentrations (1 . 5-1 . 75%) and do not express the 'transient' antigen (AGF4.36) during their maturation. The use of these lines in studies of myelopoiesis is discussed.     

Glial bridges and Schwann cell migration during chronic demyelination in the C.N.S.

Summary The formation of fibrotic bridges from subpial astrocytes into the subarachnoid space of the spinal cord and the migration of Schwann cells to the central nervous system (C.N.S.) is appraised in chronically demyelinated C.N.S. lesions. Spinal cord tissue was studied from inbred, Strain 13 guinea pigs with chronic experimental allergic encephalomyelitis (EAE). It has been found that uncommitted Schwann cells are present around remyelinated fibres in nerve root entry zones, between meningeal cells at a distance from the roots and along blood vessels within the spinal cord parenchyma. It is speculated that these cells migrate via the above route to the C.N.S. In the present model, this invasion might be aided by glial fibrosis, a process which leads to surface irregularities in the spinal cord, an extensive extracellular space and possible breaches in the glia limitans through which Schwann cells might penetrate.     

Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor

Until recently, very little was known about the molecular mechanisms responsible for the development of glaucoma, a leading cause of blindness worldwide. Mutations in the glaucoma gene myocilin (MYOC, GLC1A) are associated with elevated intraocular pressure and the development of autosomal dominant juvenile glaucoma and a subset of adult-onset glaucoma. MYOC is expressed in the trabecular meshwork (TM), a tissue responsible for drainage of aqueous humor from the eye, and the tissue involved in elevated intraocular pressure associated with glaucoma. To better understand the role of MYOC in glaucoma pathogenesis, we examined the expression of normal and mutant myocilin in cultured ocular (TM) and non-ocular cells as well as in the aqueous humor of patients with and without MYOC glaucoma. Normal myocilin was secreted from cultured cells, but very little to no myocilin was secreted from cells expressing five different mutant forms of MYOC. In addition, no mutant myocilin was detected in the aqueous humor of patients harboring a nonsense MYOC mutation (Q368X). Co-transfection of cultured cells with normal and mutant myocilin led to suppression of normal myocilin secretion. These studies suggest that MYOC glaucoma is due either to insufficient levels of secreted myocilin or to compromised TM cell function caused by congestion of the TM secretory pathway.     

Mutations in ABCA4 result in accumulation of lipofuscin before slowing of the retinoid cycle: a reappraisal of the human disease sequence

Mutations in ABCA4, which encodes a photoreceptor specific ATP-binding cassette transporter (ABCR), cause autosomal recessive forms of human blindness due to retinal degeneration (RD) including Stargardt disease. The exact disease sequence leading to photoreceptor and vision loss in ABCA4-RD is not known. Extrapolation from murine and in vitro studies predicts that two of the earliest pathophysiological features resulting from disturbed ABCR function in man would be slowed kinetics of the retinoid cycle and accelerated deposition of lipofuscin in the retinal pigment epithelium (RPE). To determine the human pathogenetic sequence, we studied surrogate measures of retinoid cycle kinetics, lipofuscin accumulation, and rod and cone photoreceptor and RPE loss in ABCA4-RD patients with a wide spectrum of disease severities. There were different extents of photoreceptor/RPE loss and lipofuscin accumulation in different regions of the retina. Slowing of retinoid cycle kinetics was not present in all patients; when present, it was not homogeneous across the retina; and the extent of slowing correlated well with the degree of degeneration. The orderly relationship between these phenotypic features permitted the development of a model of disease sequence in ABCA4-RD. The model predicted lipofuscin accumulation as a key and early component of the disease expression in man, as in mice. In man, however, abnormal slowing of the rod and cone retinoid cycle occurs at later stages of the disease sequence. Knowledge of the human ABCA4 disease sequence will be critical for defining rates of progression, selecting appropriate patients and retinal locations for future therapy, and choosing appropriate treatment outcomes.     

Evidence for linkage between regulatory enzymes in glycolysis and schizophrenia in a multiplex sample.

Observations of impaired glucose regulation in schizophrenia are long-standing, although their pathological and etiological significance is uncertain. One approach to the issue that minimizes environmental variables (e.g., medication and diet) is to determine whether genes related to glucose regulation show genetic linkage to schizophrenia. We examined the potential role of glucose metabolism in schizophrenia through a genome scan of affection status in schizophrenia and an empirical method for deriving P-values. Data were utilized from the NIMH Genetics Initiative for Schizophrenia dataset, which comprises a total sample consisting of 71 pedigrees containing 218 nuclear families and 987 individuals. A genome scan with 459 markers spaced at an average of 10 cM intervals was conducted using the linkage analysis program Genehunter separately for European- and African-American groups. Enzymes that regulate glycolysis were identified and the genes regulating these enzymes were located through the Online Mendelian Inheritance in Man (OMIM) website. The focus in this study was on genes located near previously reported schizophrenia susceptibility regions. The genome-wide significance of these genes to schizophrenia was assessed using permutation testing. When results were adjusted for multiple testing within and across ethnic groups, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2; chromosome 1q32.2) achieved genome-wide significance (P = 0.04). In addition, hexokinase 3 (HK3; chromosome 5q35.3) was also suggestive of linkage (P = 0.09). For the European-American sample, PFKFB2 (1q32.2), hexokinase 3 (HK3; 5q35.3), and pyruvate kinase 3 (PK3; chromosome 15q23) achieved significance at the 0.05 level. None of the genes showed significance in the African-American sample. Our results provide further support for the view that genes that regulate glucose metabolism may also influence susceptibility to schizophrenia. More generally, they support the view that relationships between glucose dysregulation and schizophrenia are inherent to the disorder, and are not merely epiphenomena related to medication or other treatment factors.