It has been more than 10 years since it was first proposed that the neurodegeneration in Alzheimer9s disease (AD) may be caused by deposition of amyloid β-peptide (Aβ) in plaques in brain tissue. According to the amyloid hypothesis, accumulation of Aβ in the brain is the primary influence driving AD pathogenesis. The rest of the disease process, including formation of neurofibrillary tangles containing tau protein, is proposed to result from an imbalance between Aβ production and Aβ clearance.

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The Amyloid Hypothesis of Alzheimer's Disease: Progress and Problems on the Road to Therapeutics

http://www.protocol-online.org/forums/index.php?app=core&module=attach&section=attach&attach_id=2894

Studies on transformation of Escherichia coli with plasmids

In both metazoan development and metastatic cancer, migrating cells must carry out a detailed, complex program of sensing cues, binding substrates, and moving their cytoskeletons. The linker cell in Caenorhabditis elegans males undergoes a stereotyped migration that guides gonad organogenesis, occurs with precise timing, and requires the nuclear hormone receptor NHR-67. To better understand how this occurs, we performed RNA-seq of individually staged and dissected linker cells, comparing transcriptomes from linker cells of third-stage (L3) larvae, fourth-stage (L4) larvae, and nhr-67-RNAi–treated L4 larvae. We observed expression of 8,000–10,000 genes in the linker cell, 22–25% of which were up- or down-regulated 20-fold during development by NHR-67. Of genes that we tested by RNAi, 22% (45 of 204) were required for normal shape and migration, suggesting that many NHR-67–dependent, linker cell-enriched genes play roles in this migration. One unexpected class of genes up-regulated by NHR-67 was tandem pore potassium channels, which are required for normal linker-cell migration. We also found phenotypes for genes with human orthologs but no previously described migratory function. Our results provide an extensive catalog of genes that act in a migrating cell, identify unique molecular functions involved in nematode cell migration, and suggest similar functions in humans.

https://science.sciencemag.org/content/sci/256/5054/184.full.pdf

Alzheimer's disease: the amyloid cascade hypothesis

Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.

A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping

Alzheimer's disease is characterized by a widespread functional disturbance of the human brain. Fibrillar amyloid proteins are deposited inside neurons as neurofibrillary tangles and extracellularly as amyloid plaque cores and in blood vessels. The major protein subunit (A4) of the amyloid fibril of tangles, plaques and blood vessel deposits is an insoluble, highly aggregating small polypeptide of relative molecular mass 4,500. The same polypeptide is also deposited in the brains of aged individuals with trisomy 21 (Down's syndrome). We have argued previously that the A4 protein is of neuronal origin and is the cleavage product of a larger precursor protein. To identify this precursor, we have now isolated and sequenced an apparently full-length complementary DNA clone coding for the A4 polypeptide. The predicted precursor consists of 695 residues and contains features characteristic of glycosylated cell-surface receptors. This sequence, together with the localization of its gene on chromosome 21, suggests that the cerebral amyloid deposited in Alzheimer's disease and aged Down's syndrome is caused by aberrant catabolism of a cell-surface receptor.

The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor

A set of six cloning vectors, pUR 278, 288, 289, 290, 291, 292 is presented. These vectors have the cloning sites, BamHI, SalI, PstI, XbaI and HindIII, in all frames at the 3' end of the lacZ gene. Insertion of cDNA in the proper cloning sites leads to a fusion protein of active beta-galactosidase and the peptide encoded by the cDNA. A simple immunoenzymatic assay can be used to identify clones in such a cDNA library.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1983.tb01659.x

Easy identification of cDNA clones.

The realization that the synthesis of proteins is often under the control of repressors', 2 has posed a central question in molecular biology: What is the nature of the controlling substances? The scheme of negative control proposed by Jacob and Monod envisages that certain genes, regulatory genes, make products that can act through the cytoplasm to prevent the functioning of other genes. These other genes are organized into operons with cis-dominant operators, such operators behaving as acceptors for the repressor. Appropriate small molecules act either as inducers, by preventing the repression, or as corepressors, leading to the presence of active repressor. The simplest explicit hypothesis for inducible systems is that the direct product of the control gene is itself the repressor and that this repressor binds to the operator site on a DNA molecule to prevent the transcription of the operon. The inducer would combine with the repressor to produce a molecule which can no longer bind to the operator, and the synthesis of the enzymes made by the operon would begin. However, other models will also fit the data. Repressors could have almost any target that would serve as a block to any of the initiation processes required to make a protein. A molecular understanding of the control process has waited on the isolation of one or more repressors. We have developed an assay for the lactose repressor, the product of the control gene (i gene) of the lactose operon. The assay detects and quantitates this repressor by measuring its binding to an inducer, as seen in this case by equilibrium dialysis against radioactive IPTG (isopropyl-thio-galactoside). In order to seek the lactose repressor, we desired some means that would not depend on the specific models that might be imagined for the actual mechanism of repression. The minimal assumption on which the assay is based is that there should be some interaction between the repressor and the inducer.,. However, inducing substances added to the cell are often modified before they can trigger induction. Such is the case with lactose which must be split by fl-galactosidase in order to induceI but the thiogalactosides appear to be true gratuitous inducers; no chemical modification has yet been detected associated with their action as inducers. The ability of IPTG to stabilize certain temperature-sensitive i-gene mutants4 argues strongly that the i-gene product interacts directly with this inducer. Furthermore, the existence of a competitive inhibitor of induction, ONPF (o-nitrophenylfucoside), which can also stabilize certain leaky i mutants and which behaves as though it drives the repressor into the form that shuts off the operator, also supports this thesis.5 Design of the Experiment.-In order to detect. the binding of IPTG to the repressor by equilibrium dialysis, one must achieve concentrations of repressor that are comparable to the dissociation constant of the complex. What affinity does the repressor have for the inducer? Half-maximal induction occurs at 2-10-4 M IPTG in a permeaseless strain. This fact alone does not lead directly to an estimate of the equilibrium constant because the enzyme level changes by a factor of 1,000 on

https://www.pnas.org/content/pnas/56/6/1891.full.pdf

Isolation of the lac repressor

We tested the effect of systematic destruction of all three lac operators of the chromosomal lac operon of Escherichia coli on repression by Lac repressor. Absence of just one 'pseudo-operator' O2 or O3 decreases repression by wild-type tetrameric Lac repressor approximately 2- to 3-fold; absence of both 'pseudo-operators' decreases repression greater than 50-fold. O1 alone represses under these conditions only approximately 20-fold. Dimeric active Lac repressor (iadi) represses the wild-type lac operon to about the same low extent. This indicates that cooperative interaction between lac operators is due to DNA loop formation mediated by tetrameric Lac repressor. Under conditions where loop formation is impossible, occupation of O3 but not of O2 may lead to weak repression. This suggests that under these conditions CAP activation may be inhibited and that stopping transcription at O2 does not significantly contribute to repression.

The three operators of the lac operon cooperate in repression.

The nucleotide sequence of the lacY gene coding for lactose permease (M protein) in Escherichia coli has been determined. The sequence includes the intergenic regions between the lacZ (β-galactosidase) and lacY genes as well as the region between the lacY and lacA (transacetylase) genes. Lactose permease is predicted to consist of 417 residues (71% nonpolar), resulting in a protein with a molecular weight of 46,504. The reading frame was confirmed by the sequence of a nonsense mutation changing codon 33 from UGG to UAG.

Benno Müller-Hill

Papers

The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor

Easy identification of cDNA clones.

Isolation of the lac repressor

The three operators of the lac operon cooperate in repression.

Sequence of the lactose permease gene