抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes. Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.

https://science.sciencemag.org/content/sci/270/5235/467.full.pdf

Quantitative monitoring of gene expression patterns with a complementary DNA microarray.

So far in this course we have dealt entirely with the evolution of characters that are controlled by simple Mendelian inheritance at a single locus. There are notes on the course website about gametic disequilibrium and how allele frequencies change at two loci simultaneously, but we didn’t discuss them. In every example we’ve considered we’ve imagined that we could understand something about evolution by examining the evolution of a single gene. That’s the domain of classical population genetics. For the next few weeks we’re going to be exploring a field that’s actually older than classical population genetics, although the approach we’ll be taking to it involves the use of population genetic machinery. If you know a little about the history of evolutionary biology, you may know that after the rediscovery of Mendel’s work in 1900 there was a heated debate between the “biometricians” (e.g., Galton and Pearson) and the “Mendelians” (e.g., de Vries, Correns, Bateson, and Morgan). Biometricians asserted that the really important variation in evolution didn’t follow Mendelian rules. Height, weight, skin color, and similar traits seemed to

Human biochemical genetics

We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

/pdf/gus-fusions-beta-glucuronidase-as-a-sensitive-and-versatile-4b3n5r6gfw.pdf

GUS fusions: beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

Transformed petunia, tobacco, and tomato plants have been produced by means of a novel leaf disk transformation-regeneration method. Surface-sterilized leaf disks were inoculated with an Agrobacter...

A simple and general method for transferring genes into plants

In all higher plants studied to date, the anthocyanin pigment pathway is regulated by a suite of transcription factors that include Myb, bHLH and WD-repeat proteins. However, in Arabidopsis thaliana, the Myb regulators remain to be conclusively identified, and little is known about anthocyanin pathway regulation by TTG1-dependent transcriptional complexes. Previous overexpression of the PAP1 Myb suggested that genes from the entire phenylpropanoid pathway are targets of regulation by Myb/bHLH/WD-repeat complexes in Arabidopsis, in contrast to other plants. Here we demonstrate that overexpression of Myb113 or Myb114 results in substantial increases in pigment production similar to those previously seen as a result of over-expression of PAP1, and pigment production in these overexpressors remains TTG1- and bHLH-dependent. Also, plants harboring an RNAi construct targeting PAP1 and three Myb candidates (PAP2, Myb113 and Myb114) showed downregulated Myb gene expression and obvious anthocyanin deficiencies. Correlated with these anthocyanin deficiencies is downregulation of the same late anthocyanin structural genes that are downregulated in ttg1 and bHLH anthocyanin mutants. Expression studies using GL3:GR and TTG1:GR fusions revealed direct regulation of the late biosynthetic genes only. Functional diversification between GL3 and EGL3 with regard to activation of gene targets was revealed by GL3:GR studies in single and double bHLH mutant seedlings. Expression profiles for Myb and bHLH regulators are also presented in the context of pigment production in young seedlings.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2007.03373.x

Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings.

GLABRA3 (GL3) encodes a bHLH protein that interacts with the WD repeat protein, TTG1. GL3 overexpression suppresses the trichome defect of the pleiotropic ttg1 mutations. However, single gl3 mutations only affect the trichome pathway with a modest trichome number reduction. A novel unlinked bHLH-encoding locus is described here, ENHANCER OF GLABRA3 (EGL3). When mutated, egl3 gives totally glabrous plants only in the gl3 mutant background. The double bHLH mutant, gl3 egl3, has a pleiotropic phenotype like ttg1 having defective anthocyanin production, seed coat mucilage production, and position-dependent root hair spacing. Furthermore, the triple bHLH mutant, gl3 egl3 tt8, phenocopies the ttg1 mutation. Yeast two-hybrid and plant overexpression studies show that EGL3, like GL3, interacts with TTG1, the myb proteins GL1, PAP1 and 2, CPC and TRY, and it will form heterodimers with GL3. These results suggest a combinatorial model for TTG1-dependent pathway regulation by this trio of partially functionally redundant bHLH proteins.

/pdf/a-network-of-redundant-bhlh-proteins-functions-in-all-ttg1-5a7zmunyzq.pdf

A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis.

Arabidopsis trichome development and differentiation is a well-studied model for plant cell-fate determination and morphogenesis. Mutations in TRANSPARENT TESTA GLABRA1 (TTG1) result in several pleiotropic defects including an almost complete lack of trichomes. The complex phenotype caused by ttg1 mutations is suppressed by ectopic expression of the maize anthocyanin regulator R. Here it is demonstrated that the Arabidopsis trichome development locus GLABRA3 (GL3) encodes an R homolog. GL3 and GLABRA1 (GL1) interact when overexpressed together in plants. Yeast two-hybrid assays indicate that GL3 participates in physical interactions with GL1, TTG1, and itself, but that GL1 and TTG1 do not interact. These data suggest a reiterated combinatorial model for the differential regulation of such diverse developmental pathways as trichome cell-fate determination, root hair spacing, and anthocyanin secondary metabolism.

https://www.genetics.org/content/genetics/156/3/1349.full.pdf

GL3 Encodes a bHLH Protein That Regulates Trichome Development in Arabidopsis Through Interaction With GL1 and TTG1

Glutathione S-transferases (GSTs) are enzymes that detoxify heterocyclic compounds (xenobiotics) by covalently linking glutathione to the substrate, forming a glutathione S-conjugate. A glutathione pump in the vacuolar membrane of barley actively sequesters herbicide-glutathione S-conjugates; glutathionation allows recognition and entry of the conjugates into vacuoles. The protein encoded by the Bronze-2 gene in maize performs the last genetically defined step in anthocyanin biosynthesis, resulting in the deposition of red and purple pigments in the vacuoles of maize tissues. We show here that Bz2 encodes a GST with activity in maize, transformed Arabidopsis thaliana plants and Escherichia coli. We demonstrate that anthocyanins extracted from maize protoplasts expressing BZ2 are conjugated with glutathione, and that vanadate, a known inhibitor of the glutathione pump in plant vacuolar membranes, inhibits the accumulation of anthocyanins in the vacuole. These results provide a biochemical function for BZ2, and suggest a common mechanism for the ability of plants to sequester structurally similar but functionally diverse molecules in the vacuole.

A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2.

Morphologically normal plants were regenerated from Nicotiana plumbaginifolia cells transformed with an Agrobacterium tumefaciens strain containing a tumor-inducing plasmid with a chimeric gene for kanamycin resistance. The presence of the chimeric gene in regenerated plants was demonstrated by Southern hybridization analysis, and its expression in plant tissues was confirmed by the ability of leaf segments to form callus on media containing kanamycin at concentrations that were normally inhibitory. Progeny derived from several transformed plants inherited the foreign gene in a Mendelian manner.

https://science.sciencemag.org/content/sci/223/4635/496.full.pdf

Alan M Lloyd

Papers

Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings.

A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis.

GL3 Encodes a bHLH Protein That Regulates Trichome Development in Arabidopsis Through Interaction With GL1 and TTG1

A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2.

Inheritance of functional foreign genes in plants.