Microsatellite allele frequencies in humans and chimpanzees, with implications for constraints on allele size

The distributions of allele sizes at eight simple-sequence repeat (SSR) or microsatellite loci in chimpanzees are found and compared with the distributions previously obtained from several human populations. At several loci, the differences in average allele size between chimpanzees and humans are sufficiently small that there might be a constraint on the evolution of average allele size. Furthermore, a model that allows for a bias in the mutation process shows that for some loci a weak bias can account for the observations. Several alleles at one of the loci (Mfd 59) were sequenced. Differences between alleles of different lengths were found to be more complex than previously assumed. An 8-base-pair deletion was present in the nonvariable region of the chimpanzee locus. This locus contains a previously unrecognized repeated region, which is imperfect in humans and perfect in chimpanzees. The apparently greater opportunity for mutation conferred by the two perfect repeat regions in chimpanzees is reflected in the higher variance in repeat number at Mfd 59 in chimpanzees than in humans. These data indicate that interspecific differences in allele length are not always attributable to simple changes in the number of repeats.      

The program XEASY for computer-supported NMR spectral analysis of biological macromolecules

Summary A new program package, XEASY, was written for interactive computer support of the analysis of NMR spectra for three-dimensional structure determination of biological macromolecules. XEASY was developed for work with 2D, 3D and 4D NMR data sets. It includes all the functions performed by the precursor program EASY, which was designed for the analysis of 2D NMR spectra, i.e., peak picking and support of sequence-specific resonance assignments, cross-peak assignments, cross-peak integration and rate constant determination for dynamic processes. Since the program utilizes the X-window system and the Motif widget set, it is portable on a wide range of UNIX workstations. The design objective was to provide maximal computer support for the analysis of spectra, while providing the user with complete control over the final resonance assignments. Technically important features of XEASY are the use and flexible visual display of strips, i.e., two-dimensional spectral regions that contain the relevant parts of 3D or 4D NMR spectra, automated sorting routines to narrow down the selection of strips that need to be interactively considered in a particular assignment step, a protocol of resonance assignments that can be used for reliable bookkeeping, independent of the assignment strategy used, and capabilities for proper treatment of spectral folding and efficient transfer of resonance assignments between spectra of different types and different dimensionality, including projected, reduced-dimensionality triple-resonance experiments.Abbreviations 1D, 2D, 3D, 4D one-, two-, three-, four-dimensional - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy - COSY correlation spectroscopy - TPPI time-proportional phase incrementation     

Rescue of measles viruses from cloned DNA.

A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome.     

Functional and nonfunctional measles virus matrix genes from lethal human brain infections.

Subacute sclerosing panencephalitis (SSPE) is a lethal disease induced by the persistence of measles virus in the human brain. In many SSPE cases, the viral matrix (M) protein cannot be detected; in others, M proteins of the expected size are found and sequence analysis of M cDNAs has confirmed that the reading frames are intact, showing only several missense mutations. To determine whether these alterations result in nonfunctional proteins, we have replaced the M gene of an infectious full-length genomic cDNA (from vaccine strain Edmonston) with different M genes derived from four patients with SSPE. One of the SSPE M genes tested proved to be functionally competent, giving rise to a virus yielding titers similar to those of viruses containing the M gene from control lytic strains. The other three SSPE M genes were not functionally competent in the same test. In all three cases, the inactivating changes resided in the carboxyl-terminal half of the M protein, as shown by the exchange of either of the two genes halves. In summary, mutational M gene alterations, which either prevent synthesis of M protein altogether or only allow synthesis of nonfunctional M protein, have been detected by us and by others in 9 of 10 SSPE cases. The one functional M gene appears to be an exception to the rule, indicating that M gene alteration might not be an absolute requirement for disease development.     

The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution: comparison with prokaryotic repressors

The structure of the Antennapedia homeodomain from Drosophila melanogaster was determined by nuclear magnetic resonance spectroscopy in solution. It includes three well-defined helices (residues 10-21, 28-38, and 42-52) and a more flexible fourth helix (residues 53-59). Residues 30-50 form a helix-turn-helix motif virtually identical to those observed in various prokaryotic repressors. Further comparisons of the homeodomain with prokaryotic repressors showed that there are also significant differences in the molecular architectures. Overall, these studies support the view that the third helix of the homeodomain may function as the DNA recognition site. The elongation of the third helix by the fourth helix is a structured element that so far appears to be unique to the Antennapedia homeodomain.     

Fucosylated protein of retinal cone photoreceptor outer segments: morphological and biochemical analyses

Cone outer segments (OS) of the goldfish retina are diffusely labeled after intravitreal injection of [(3)H]fucose while rod OS remain unlabeled. By electron microscopic radioautography, the OS of red- and blue-sensitive cones are heavily labeled while green- sensitive cone OS are lightly labeled. The time-course and pattern of OS labeling in all cone types from 30 min to 24 h resemble that of incorporation of other sugars into rhodopsin in rod OS. The nature of the cone OS-specific fucosylated component(s) was examined using biochemical techniques. Cone OS were prelabeled by intravitreal injection of [(3)H]fucose 24 h before sacrifice. Photoreceptor OS were isolated using a discontinuous sucrose density gradient and it was verified by electron microscopic radioautography that the only source of radioactivity in the preparations was cone OS. The different cone types could be recognized by the heaviness of labeling, characteristic membrane spacing, and 'staining' of green cone OS in vitro with horseradish peroxidase. After acid hydrolysis of prelabeled photoreceptor membranes, 90 percent of the counts were in the neutral sugar fraction which was analyzed by thin-layer chromatography. Approximately 70 percent of the radioactivity co-chromatographed with authentic fucose. SDS-PAGE/fluorography of prelabeled photoreceptor membranes revealed a single radioactive component that was lightly stained with coomassie blue and showed an apparent molecular weight of 33,000. This cone-derived band was separated from unlabeled rod opsin which was well stained and showed an apparent mol wt of 38,000. Isoelectric focusing under denaturing conditions produced two major and one minor band of radioactivity with isoelectric points of 8.2, 8.6, and 8.8 respectively. No radioactivity was found in association with a stained band corresponding in isoelectric point to that of bovine opsin (pl, 6.2). The fucosylated component was readily digested by pronase, indicating its protein nature. Washing of the isolated OS with isotonic and hypotonic buffers failed to extract major amounts of the radioactivity, suggesting that the fucosylated component is an integral membrane protein. The presence of a fucosylated protein thus represents a major difference between cone and rod OS in the goldfish and has enabled us to identify cone OS in preparations of isolated photoreceptor membranes and to demonstrate the separation of a cone-derived glycoprotein from rod opsin.     

Characterization of the 3'-terminal region of the large molecular weight RNA subunits from normal and transformation-defective Rous sarcoma virus.

In avian sarcoma viruses a function required to transform cells is frequently lost giving rise to non-transforming virus. The genetic information for this function is localized near the 3' end of the genome. Short 3'-terminal poly(A)-linked RNA fragments from the genomes of a transforming Rous sarcoma virus and its non-transforming derivative were isolated and gave rise to identical fingerprint patterns, suggesting that internal deletion rather than terminal elimination leads to non-transforming virus.