Last year, whilst going through lecture notes for the BMB and CDB courses at Cambridge, I decided to collate the comments about things that were not known.
The result is this list of some of the things we don’t know about biochemisty/molecular biology/cell biology.
Each bullet point is a direct quotation from lecture notes; comments in square brackets were added by me as explanation.
###Things ‘Unknown’: * Analysis of Arabidopsis genome sequence identified 450 genes for PPR proteins – all predicted to be targeted to chloroplasts or mitochondria. Roles of most unknown, but some shown to be important for splicing, translation, and editing of chloroplast transcripts.
b559 is cytochrome of unknown function.
However, cloning of the receptor revealed a thrombin-cleavage site close to the N terminus, and functional studies showed that thrombin clipped off a short peptide (whose function is unknown), revealing a new N-terminus with the sequence Ser-Phe-Leu-Leu-Arg—-. This peptide binds to the receptor, activating it conventionally.
The location of the secretion “signal” in the structurally-diverse proteins targeted through the Type II pathway is unknown.
The default state for var genes is to be silenced; this property is conferred by telomeric location and by an unknown mechanism involving sequences in the intron and exon 2 and probably involves chromatin remodelling during replication
A common way of making E. coli competent is to treat them with CaCl2, which works by an unknown mechanism to permit them to take up DNA when they are subjected to a heat shock.
The detailed mechanism of plasmid partition is unknown but some years ago a plausible scenario was described in the pre-pairing model (Fig. 12) which proposes that a monomeric protein binds to a centromere-like site on individual plasmids.
In any case, selective retrograde transport by COPI vesicles must play an import role for intra-Golgi sorting; the necessary sorting signals however are still largely unknown.
Later in development, the GLP-l protein is involved a second time in signalling that is required for anterior pharynx formation. GLP-l acts as the receptor on the ABa derived cells for the unknown ligand from the MS cell.
Live cell imaging shows that this secretion is mediated by tubular carriers, which are “pulled” out of the TGN by molecular motors. Surprisingly, these carriers have not been characterised in detail, and it is still not known what protein coat (if any) they require.
The basis for the different affinities [of Erd2 for its cargo] in the ER and ERGIC is not known; it seems plausible that differences in pH and ionic environment could be accountable.
Cloning strategies have allowed a large number of novel receptors to be identified as orphans, i.e. their ligand is not known.
The role of SRC in the normal cell cycle is not known
The active component of the germ plasm is not known, but it is interesting that the germ plasm contains the homologues of some genes that are expressed in the pole plasm of Drosophila
Use of a dominant-negative receptor shows that activin-like signalling is required for normal mesoderm formation, but it is not known which of the above factors is most important in this process.
What is the molecular nature of the trigger that initiates the activation of Cdk1. The precise nature of the trigger is not known but it is believed it is centred on the spindle pole body/centrosome.
Other types of activation domain have more recently been discovered (“glutamine-rich”; “proline-rich); significance unclear.
Whether both branches [of PS1] are active is unclear.
Kinetochore MTs shorten by the net loss of tubulin from their kinetochore ends. Yet, how MT depolymerization at the kinetochore drives chromosome movement (without MTs letting go of the kinetochores!) remains unclear.
Inactivation of AS160 in response to insulin signalling results in increased Rab GTP loading and thus allows translocation to the plasma membrane. However the exact mechanism remains unclear.
The actual composition of the pore is controversial and still unclear, but a key component is probably the adenine nucleotide translocator, which forms a pore when high levels of calcium bind to the matrix side of the translocator.
Very recent genomic research provides accumulating evidence for many more narrowly defined origins and initiation zones, many of which are located in the vicinity of active gene promoters. However, the genetic and epigenetic factors that determine an active origin, or that determine its activation timing are still unclear.
Brefeldin is a very useful drug for disrupting Golgi apparatus integrity, even though it is not clear what physiological significance the Golgi-derived tubules have.
Not clear whether it [Z DNA] exists in vivo
In solution, DNA is usually B-form and this is the major form in the cell. Dehydration (in fibres, many crystals) produces A-form; not clear whether any significant stretches of A-DNA exist in vivo (although RNA-DNA hybrids are A-form).
In summary, in eukaryotes supercoiling could be generated locally by transcription. Not clear that there is a role for it in driving transcription, e.g. by promoter unwinding.
The H+/2e- stoichiometry of complex I is 4, but the mechanism of proton coupling is not clear.
However the causes and mechanistic basis of type 2 diabetes are not clear.
Curiously, the active sites [in TIM barrel proteins] are found at the C-‐termini of the β-‐strands and N-‐termini of the helices; it is not clear that this can always be explained in terms of helix dipoles or divergent evolution.
Several receptor specific kinases are known, but it is not clear whether all 7TM receptors are susceptible to their action.
###Things ‘Not Understood’: * Sorting into regulated secretory vesicles is not understood in detail. It is likely to involve kin recognition, as well as retrograde sorting of unwanted proteins from budded immature secretory vesicles.
It has also been proposed that RI- and RII-containing isoforms of PKA may be regulated by different ligands in the same cell type, implying that these may be expressed in different compartments within the cell. The basis for this remains to be established.
The relationship between IP4 and Ca2+ entry remains contentious after many years.
PI-3 K can also phosphorylate PI and PIP at the 3 position, generating novel isoforms of PIP and PIP2. Whether these lipids serve a signalling function remains to be seen.
The regulation of cPLA2 remains unclear.
It is interesting that carbon monoxide (CO) appears to couple to GC in the same way. (This might be anticipated, given the known binding of CO to haem in haemoglobin.) Any physiological significance of the action of CO remains to be established.
Whether the fly centromere is a good model for other eukaryotic centromeres remains to be seen.