Identification of distinct T cell receptor (TCR)-gamma delta heterodimers using an anti-TCR-gamma variable region serum

T cell hybridomas were generated from CD3+, CD4-, CD8- splenocytes and fetal thymocytes. V gamma 1-expressing proteins present on these murine TCR-gamma delta hybridomas were identified by using an anti-TCR V gamma 1 peptide serum. This antiserum specifically immunoprecipitated 41-kDa TCR V gamma-C gamma 4 chains and 31-kDa TCR V gamma-C gamma 1/2 chains from distinct heterodimers expressed on the TCR-gamma delta T cell hybridomas. The RNA from a hybridoma with a 31-kDa TCR-gamma chain hybridized with a V gamma 1 probe but failed to hybridize with a V gamma 2 probe. In contrast, the RNA from a hybridoma with a 32-kDa TCR-gamma chain hybridized with a V gamma 2 probe. This 32-kDa TCR-gamma chain was not immunoprecipitated by the anti-V gamma 1 serum. These data were consistent with the conclusion that the 31-kDa protein was the product of a V gamma 1 to C gamma 2 rearrangement, whereas the 32-kDa protein was the product of a V gamma 2 to C gamma 1 rearrangement. Furthermore, Southern analyses confirmed that the 32-kDa protein was the product of a V gamma 1.2-J gamma 2 rearrangement, and all three of the 41-kDa TCR-gamma chains were the results of V gamma 1.1-J gamma 4 rearrangements. This was the first demonstration at the clonal level of TCR-gamma proteins which use members of the V gamma 1 gene family, as well as the C gamma 2 constant region. Additional biochemical analyses of the TCR-gamma and -delta proteins from three independently derived C gamma 4-bearing T cell hybridomas suggested that most of the molecular mass diversity observed in the bulk subpopulation of peripheral C gamma 4-containing heterodimers may be contributed by the TCR-delta chains.     

Uniparental isodisomy of chromosome 2 causing MRPL44-related multisystem mitochondrial disease

Mutations in nuclear-encoded protein subunits of the mitochondrial ribosome are an increasingly recognised cause of oxidative phosphorylation system (OXPHOS) disorders. Among them, mutations in the MRPL44 gene, encoding a structural protein of the large subunit of the mitochondrial ribosome, have been identified in four patients with OXPHOS defects and early-onset hypertrophic cardiomyopathy with or without additional clinical features. A 23-year-old individual with cardiac and skeletal myopathy, neurological involvement, and combined deficiency of OXPHOS complexes in skeletal muscle was clinically and genetically investigated. Analysis of whole-exome sequencing data revealed a homozygous mutation in MRPL44 (c.467 T?>?G), which was not present in the biological father, and a region of homozygosity involving most of chromosome 2, raising the possibility of uniparental disomy. Short-tandem repeat and genome-wide SNP microarray analyses of the family trio confirmed complete maternal uniparental isodisomy of chromosome 2. Mitochondrial ribosome assembly and mitochondrial translation were assessed in patient derived-fibroblasts. These studies confirmed that c.467 T?>?G affects the stability or assembly of the large subunit of the mitochondrial ribosome, leading to impaired mitochondrial protein synthesis and decreased levels of multiple OXPHOS components. This study provides evidence of complete maternal uniparental isodisomy of chromosome 2 in a patient with MRPL44-related disease, and confirms that MRLP44 mutations cause a mitochondrial translation defect that may present as a multisystem disorder with neurological involvement.

     

Phylogeographical population structure of tiger quolls Dasyurus maculatus (Dasyuridae: Marsupialia), an endangered carnivorous marsupial

Tiger quolls, Dasyurus maculatus, are the largest carnivorous marsupials still extant on the mainland of Australia, and occupy an important ecological niche as top predators and scavengers. Two allopatric subspecies are recognized, D.m. gracilis in north Queensland, and D.m. maculatus in the southeast of the mainland and Tasmania. D.m. gracilis is considered endangered while D.m. maculatus is listed as vulnerable to extinction; both subspecies are still in decline. Phylogeographical subdivision was examined to determine evolutionarily significant units (ESUs) and management units (MUs) among populations of tiger quolls to assist in the conservation of these taxa. Ninety-three tiger quolls from nine representative populations were sampled from throughout the species range. Six nuclear microsatellite loci and the mitochondrial DNA (mtDNA) control region (471 bp) were used to examine ESUs and MUs in this species. We demonstrated that Tasmanian tiger quolls are reciprocally monophyletic to those from the mainland using mtDNA analysis, but D.m. gracilis was not monophyletic with respect to mainland D.m. maculatus. Analysis of microsatellite loci also revealed significant differences between the Tasmanian and mainland tiger quolls, and between D.m. gracilis and mainland D.m. maculatus. These results indicate that Tasmanian and mainland tiger quolls form two distinct evolutionary units but that D.m. gracilis and mainland D.m. maculatus are different MUs within the same ESU. The two marker types used in this study revealed different male and female dispersal patterns and indicate that the most appropriate units for short-term management are local populations. A revised classification and management plan are needed for tiger quolls, particularly in relation to conservation of the Tasmanian and Queensland populations.     

Gestational length in the koala, Phascolarctos cinereus

We report a possible case of extended gestation in the koala, Phascolarctos cinereus. Birth of a pouch young was first observed 127 days after the removal of the male from a multi-female colony at Taronga Zoo. No other males were present at that time or had access to the facility. Head measurements and other growth data collected at the time of detection and over the period of pouch life indicates the time from removal of the male and the date of birth to be between 50 and 77 days. DNA fingerprinting using microsatellite loci unambiguously assigned paternity of the pouch young to this male.

These observations suggest either an extended period of gestation of at least 50 days, or activation of a dormant blastocyst from the previous breeding season, as the female entered the period of seasonal oestrus.     

Characterisation and Validation of Insertions and Deletions in 173 Patient Exomes

Recent advances in genomics technologies have spurred unprecedented efforts in genome and exome re-sequencing aiming to unravel the genetic component of rare and complex disorders. While in rare disorders this allowed the identification of novel causal genes, the missing heritability paradox in complex diseases remains so far elusive. Despite rapid advances of next-generation sequencing, both the technology and the analysis of the data it produces are in its infancy. At present there is abundant knowledge pertaining to the role of rare single nucleotide variants (SNVs) in rare disorders and of common SNVs in common disorders. Although the 1,000 genome project has clearly highlighted the prevalence of rare variants and more complex variants (e.g. insertions, deletions), their role in disease is as yet far from elucidated.We set out to analyse the properties of sequence variants identified in a comprehensive collection of exome re-sequencing studies performed on samples from patients affected by a broad range of complex and rare diseases (N = 173). Given the known potential for Loss of Function (LoF) variants to be false positive, we performed an extensive validation of the common, rare and private LoF variants identified, which indicated that most of the private and rare variants identified were indeed true, while common novel variants had a significantly higher false positive rate. Our results indicated a strong enrichment of very low-frequency insertion/deletion variants, so far under-investigated, which might be difficult to capture with low coverage and imputation approaches and for which most of study designs would be under-powered. These insertions and deletions might play a significant role in disease genetics, contributing specifically to the underlining rare and private variation predicted to be discovered through next generation sequencing.     

Influence of plant developmental stage on microbial community structure and activity in the rhizosphere of three field crops

Seasonal shifts in rhizosphere microbial populations were investigated to follow the influence of plant developmental stage. A field study of indigenous microbial rhizosphere communities was undertaken on pea (Pisum satvium var. quincy), wheat (Triticum aestivum var. pena wawa) and sugar beet (Beta vulgaris var. amythyst). Rhizosphere community diversity and substrate utilization patterns were followed throughout a growing season, by culturing, rRNA gene density gradient gel electrophoresis and BIOLOG. Culturable bacterial and fungal rhizosphere community densities were stable in pea and wheat rhizospheres, with dynamic shifts observed in the sugar beet rhizosphere. Successional shifts in bacterial and fungal diversity as plants mature demonstrated that different plants select and define their own functional rhizosphere communities. Assessment of metabolic activity and resource utilization by bacterial community-level physiological profiling demonstrated greater similarities between different plant species rhizosphere communities at the same than at different developmental stages. Marked temporal shifts in diversity and relative activity were observed in rhizosphere bacterial communities with developmental stage for all plant species studied. Shifts in the diversity of fungal and bacterial communities were more pronounced in maturing pea and sugar beet plants. This detailed study demonstrates that plant species select for specialized microbial communities that change in response to plant growth and plant inputs.