Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)_n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.     

Spontaneous allelic variant in deafness–blindness gene Ush1g resulting in an expanded phenotype

Relationships between novel phenotypic behaviors and specific genetic alterations are often discovered using target-specific, directed mutagenesis or phenotypic selection following chemical mutagenesis. An alternative approach is to exploit deficiencies in DNA repair pathways that maintain genetic integrity in response to spontaneously induced damage. Mice deficient in the DNA glycosylase NEIL1 show elevated spontaneous mutations, which arise from translesion DNA synthesis past oxidatively induced base damage. Several litters of Neil1 knockout mice included animals that were distinguished by their backwards-walking behavior in open-field environments, while maintaining frantic forward movements in their home cage environment. Other phenotypic manifestations included swim test failures, head tilting and circling. Mapping of the mutation that conferred these behaviors showed the introduction of a stop codon at amino acid 4 of the Ush1g gene. Ush1g^bw/bw null mice displayed auditory and vestibular defects that are commonly seen with mutations affecting inner-ear hair-cell function, including a complete lack of auditory brainstem responses and vestibular-evoked potentials. As in other Usher syndrome type I mutant mouse lines, hair cell phenotypes included disorganized and split hair bundles, as well as altered distribution of proteins for stereocilia that localize to the tips of row 1 or row 2. Disruption to the bundle and kinocilium displacement suggested that USH1G is essential for forming the hair cell's kinocilial links. Consistent with other Usher type 1 models, Ush1g^bw/bw mice had no substantial retinal degeneration compared with Ush1g^bw/+ controls. In contrast to previously described Ush1g alleles, this new allele provides the first knockout model for this gene.     

Reflective organelles in the anterior pigment epithelium of the iris of the European starling Sturnus vulgaris

The ultrastructure and chemical composition of reflective organelles in the anterior pigment epithelium of the iris of the European starling Sturnus vulgaris were examined. The reflective organelles produced a diffuse white reflectance at the iris mid-section which was visible only when the stroma was removed. The pigment granules were clear, angular, and birefringent under the light microscope. In electron micrographs the granules were irregular in shape and density, sometimes crystalline in appearance, but more often they were lost during sectioning or staining. Guanine was abundant in the modified pigment epithelium of the starling, but not in the pigment epithelia of other birds that lacked birefringent granules. Pteridines, such as xanthopterin and leucopterin, were present in small amounts. Pteridines were also present in the iris stroma which had no reflective organelles. The reflective organelles in the starling pigment epithelium resemble both the reflecting platelets of lower vertebrate chromatophores and the reflective granules in the tapeta of various vertebrates. Possible derivation of the organelles from these sources is discussed.     

Purification and Characterization of an Autolysin from Clostridium acetobutylicum

A proteinaceous substance with antibiotic-like activity, resembling that of a bacteriocin, was isolated from an industrial-scale acetone-butanol fermentation of Clostridium acetobutylicum. The substance, purified by acetone precipitation, diethylaminoethyl cellulose chromatography, and polyacrylamide gel electrophoresis, was characterized as a glycoprotein with a molecular weight of 28,000. The glycoprotein was partially inactivated by certain protease enzymes. It had no effect on deoxyribonucleic acid, ribonucleic acid, or protein synthesis, and it did not result in the loss of intracellular adenosine triphosphate. The glycoprotein lysed sodium dodecyl sulfate-treated cells and cell wall preparations, and therefore it is referred to as an autolysin. The autolysin gene appeared to be chromosomal since plasmid deoxyribonucleic acid was not detected in the C. acetobutylicum strain.     

Development and validation of genetic markers for sex and cannabinoid chemotype in Cannabis sativa L.

Hemp (Cannabis sativa L.) is an emerging dioecious crop grown primarily for grain, fiber, and cannabinoids. There is good evidence for medicinal benefits of the most abundant cannabinoid in hemp, cannabidiol (CBD). For CBD production, female plants producing CBD but not tetrahydrocannabinol (THC) are desired. We developed and validated high‐throughput PACE (PCR Allele Competitive Extension) assays for C. sativa plant sex and cannabinoid chemotype. The sex assay was validated across a wide range of germplasm and resolved male plants from female and monoecious plants. The cannabinoid chemotype assay revealed segregation in hemp populations, and resolved plants producing predominantly THC, predominantly CBD, and roughly equal amounts of THC and CBD. Cultivar populations that were thought to be stabilized for CBD production were found to be segregating phenotypically and genotypically. Many plants predominantly producing CBD accumulated more than the current US legal limit of 0.3% THC by dry weight. These assays and data provide potentially useful tools for breeding and early selection of hemp.     

An ATP Binding Cassette Transporter Is Required for Cuticular Wax Deposition and Desiccation Tolerance in the Moss Physcomitrella patens

The plant cuticle is thought to be a critical evolutionary adaptation that allowed the first plants to colonize land, because of its key roles in regulating plant water status and providing protection from biotic and abiotic stresses. Much has been learned about cuticle composition and structure through genetic and biochemical studies of angiosperms, as well as underlying genetic pathways, but little is known about the cuticles of early diverging plant lineages. Here, we demonstrate that the moss Physcomitrella patens, an extant relative of the earliest terrestrial plants, has a cuticle that is analogous in both structure and chemical composition to those of angiosperms. To test whether the underlying cuticle biosynthetic pathways were also shared among distant plant lineages, we generated a genetic knockout of the moss ATP binding cassette subfamily G (ABCG) transporter Pp-ABCG7, a putative ortholog of Arabidopsis thaliana ABCG transporters involved in cuticle precursor trafficking. We show that this mutant is severely deficient in cuticular wax accumulation and has a reduced tolerance of desiccation stress compared with the wild type. This work provides evidence that the cuticle was an adaptive feature present in the first terrestrial plants and that the genes involved in their formation have been functionally conserved for over 450 million years.