Factors affecting somatic embryogenesis and transformation in modern plant breeding

Somatic embryogenesis and transformation systems are indispensable modern plant breeding components since they provide an alternative platform to develop control strategies against the plethora of pests and diseases affecting many agronomic crops. This review discusses some of the factors affecting somatic embryogenesis and transformation, highlights the advantages and limitations of these systems and explores these systems as breeding tools for the development of crops with improved agronomic traits. The regeneration of non-chimeric transgenic crops through somatic embryogenesis with introduced disease and pest-resistant genes for instance, would be of significant benefit to growers worldwide.

Frequent activating FGFR2 mutations in endometrial carcinomas parallel germline mutations associated with craniosynostosis and skeletal dysplasia syndromes

Endometrial carcinoma is the most common gynecological malignancy in the United States. Although most women present with early disease confined to the uterus, the majority of persistent or recurrent tumors are refractory to current chemotherapies. We have identified a total of 11 different FGFR2 mutations in 3/10 (30%) of endometrial cell lines and 19/187 (10%) of primary uterine tumors. Mutations were seen primarily in tumors of the endometrioid histologic subtype (18/115 cases investigated, 16%). The majority of the somatic mutations identified were identical to germline activating mutations in FGFR2 and FGFR3 that cause Apert Syndrome, Beare-Stevenson Syndrome, hypochondroplasia, achondroplasia and SADDAN syndrome. The two most common somatic mutations identified were S252W (in eight tumors) and N550K (in five samples). Four novel mutations were identified, three of which are also likely to result in receptor gain-of-function. Extensive functional analyses have already been performed on many of these mutations, demonstrating they result in receptor activation through a variety of mechanisms. The discovery of activating FGFR2 mutations in endometrial carcinoma raises the possibility of employing anti-FGFR molecularly targeted therapies in patients with advanced or recurrent endometrial carcinoma.

The CDKN2A (p16) gene and human cancer

CDKN2A, the gene encoding the cell-cycle inhibitor p16CDKN2A, was first identified in 1994. Since then, somatic mutations have been observed in many cancers and germline alterations have been found in kindreds with familial atypical multiple mole/melanoma (FAMMM), also known as atypical mole syndrome. In this review we tabulate the known mutations in this gene and discuss specific aspects, particularly with respect to germline mutations and cancer predisposition.

CDKN2A mutation in a non-FAMMM kindred with cancers at multiple sites results in a functionally abnormal protein

The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations.

Mutation analysis of the CDKN2A promoter in Australian melanoma families

Approximately 50% of all melanoma families worldwide show linkage to 9p21-22, but only about half of these have been shown to contain germ line CDKN2A mutations. It has been hypothesized that a proportion of these families carry mutations in the noncoding regions of CDKN2A. Several Canadian families have been reported to carry a mutation in the 5' UTR, at position -34 relative to the start site, which gives rise to a novel AUG translation initiation codon that markedly decreases translation from the wild-type AUG (Liu et al., 1999). Haplotype sharing in these Canadian families suggested that this mutation is of British origin. We sequenced 1,327 base pairs (bp) of CDKN2A, making up 1,116 bp of the 5' UTR and promoter, all of exon 1, and 61 bp of intron 1, in at least one melanoma case from 110 Australian families with three or more affected members known not to carry mutations within the p16 coding region. In addition, 431 bp upstream of the start codon was sequenced in an additional 253 affected probands from two-case melanoma families for which the CDKN2A mutation status was unknown. Several known polymorphisms at positions -33, -191, -493, and -735 were detected, in addition to four novel variants at positions 120, -252, -347, and -981 relative to the start codon. One of the probands from a two-case family was found to have the previously reported Q50R mutation. No family member was found to carry the mutation at position -34 or any other disease-associated mutation. For further investigation of noncoding CDKN2A mutations that may affect transcription, allele-specific expression analysis was carried out in 31 of the families with at least three affected members who showed either complete or "indeterminate" 9p haplotype sharing without CDKN2A exonic mutations. Reverse transcription polymerase chain reaction and automated sequencing showed expression of both CDKN2A alleles in all family members tested. The lack of CDKN2A promoter mutations and the absence of transcriptional silencing in the germ line of this cohort of families suggest that mutations in the promoter and 5' UTR play a very limited role in melanoma predisposition.

A genome-based strategy uncovers frequent BRAF mutations in melanoma

Using a genome-scanning approach to search for oncogenes, a recent report identifies somatic mutations in the signaling gene BRAF that are particularly prevalent in melanoma.

PTEN inactivation is rare in melanoma tumours but occurs frequently in melanoma cell lines

Deletions detected in cytogenetic and loss of heterozygosity (LOH) studies indicate that at least one tumour suppressor gene maps to the long arm of chromosome 10. Previous deletion mapping studies have observed LOH on 10q in about 30% of melanomas analysed. The PTEN gene, mapping to chromosome band 10q23.3, encodes a protein with both lipid and protein phosphatase activity. Somatic mutations and deletions in have been detected in a variety of cell lines and tumours, including melanoma samples. We performed mutation analyses and extensive allelic loss studies to investigate the role this gene plays in melanoma pathogenesis. We found that a total of 34 out of 57 (60%) melanoma cell lines carried hemizygous deletions of chromosome 10q encompassing the PTEN locus. A further three cell lines carried smaller deletions excluding PTEN. Inactivation of both PTEN alleles by exon-specific homozygous deletion or mutation was observed in 13 out of 57 (23%) melanoma cell lines. The mutation spectrum observed does not indicate an important role for ultraviolet radiation in the genesis of these mutations, and evidence from three cell lines supports the acquisition of PTEN aberrations in culture. Ten out of 49 (20%) matched melanoma tumour/normal samples harboured hemizygous deletions of either the whole chromosome or most of the long arm. Mutations within were detected in only one of the 10 tumours demonstrating LOH at 10q23 that were analysed. These results suggest that PTEN inactivation may be important for the propagation of melanoma cells in culture, and that another chromosome 10 tumour suppressor gene may be important for melanoma pathogenesis.

Microarray expression profiling in melanoma reveals a BRAF mutation signature

We have used microarray gene expression profiling and machine learning to predict the presence of BRAF mutations in a panel of 61 melanoma cell lines. The BRAF gene was found to be mutated in 42 samples (69%) and intragenic mutations of the NRAS gene were detected in seven samples (11%). No cell line carried mutations of both genes. Using support vector machines, we have built a classifier that differentiates between melanoma cell lines based on BRAF mutation status. As few as 83 genes are able to discriminate between BRAF mutant and BRAF wild-type samples with clear separation observed using hierarchical clustering. Multidimensional scaling was used to visualize the relationship between a BRAF mutation signature and that of a generalized mitogen-activated protein kinase (MAPK) activation (either BRAF or NRAS mutation) in the context of the discriminating gene list. We observed that samples carrying NRAS mutations lie somewhere between those with or without BRAF mutations. These observations suggest that there are gene-specific mutation signals in addition to a common MAPK activation that result from the pleiotropic effects of either BRAF or NRAS on other signaling pathways, leading to measurably different transcriptional changes.

Methods used in cross-cultural comparisons of somatic symptoms and their determinants

This paper is the third in a series of reviews of cross-cultural studies of symptoms at midlife. The goal of this review is to examine methods used previously in cross-cultural studies of menopause and women's health at midlife to (1) identify challenges in the measurement of somatic symptoms across cultures and (2) recommend questions and tools that can be used in future research. This review also aims to examine the determinants of somatic symptoms. The review concludes that methods used for assessing somatic symptoms differ across studies. Somatic symptoms, particularly, aches, pain, and fatigue have a high prevalence. Statistically significant differences were seen in the prevalence of somatic symptoms across cultures. Based on the number of studies that demonstrated cross-cultural differences in symptom prevalence, we recommend that the following symptoms be included in future studies of symptoms at midlife: headaches, aches/pain, palpitations, dizziness, fatigue, breathing difficulties, numbness or tingling, and gastrointestinal difficulties. We also recommend that objective measures of physical function be administered when possible to supplement subjective self-evaluation.

A boundary preserving numerical algorithm for the Wright-Fisher model with mutation

The Wright-Fisher model is an Itô stochastic differential equation that was originally introduced to model genetic drift within finite populations and has recently been used as an approximation to ion channel dynamics within cardiac and neuronal cells. While analytic solutions to this equation remain within the interval [0,1], current numerical methods are unable to preserve such boundaries in the approximation. We present a new numerical method that guarantees approximations to a form of Wright-Fisher model, which includes mutation, remain within [0,1] for all time with probability one. Strong convergence of the method is proved and numerical experiments suggest that this new scheme converges with strong order 1/2. Extending this method to a multidimensional case, numerical tests suggest that the algorithm still converges strongly with order 1/2. Finally, numerical solutions obtained using this new method are compared to those obtained using the Euler-Maruyama method where the Wiener increment is resampled to ensure solutions remain within [0,1].

Drugs and the somatic nervous system

Drugs and the somatic nervous system 8.1 The somatic nervous system 8.2 Anticholinesterases 8.3 Neuromuscular blockers 8.4 Botox

A three-nucleotide mutation altering the Maize streak virus Rep pRBR-interaction motif reduces symptom severity in maize and partially reverts at high frequency without restoring pRBR-Rep binding

Geminivirus infectivity is thought to depend on interactions between the virus replication-associated proteins Rep or RepA and host retinoblastoma-related proteins (pRBR), which control cell-cycle progression. It was determined that the substitution of two amino acids in the Maize streak virus (MSV) RepA pRBR-interaction motif (LLCNE to LLCLK) abolished detectable RepA-pRBR interaction in yeast without abolishing infectivity in maize. Although the mutant virus was infectious in maize, it induced less severe symptoms than the wild-type virus. Sequence analysis of progeny viral DNA isolated from infected maize enabled detection of a high-frequency single-nucleotide reversion of C(601)A in the 3 nt mutated sequence of the Rep gene. Although it did not restore RepA-pRBR interaction in yeast, sequence-specific PCR showed that, in five out of eight plants, the C(601)A reversion appeared by day 10 post-inoculation. In all plants, the C(601)A revertant eventually completely replaced the original mutant population, indicating a high selection pressure for the single-nucleotide reversion. Apart from potentially revealing an alternative or possibly additional function for the stretch of DNA that encodes the apparently non-essential pRBR-interaction motif of MSV Rep, the consistent emergence and eventual dominance of the C(601)A revertant population might provide a useful tool for investigating aspects of MSV biology, such as replication, mutation and evolution rates, and complex population phenomena, such as competition between quasispecies and population turnover. © 2005 SGM.