dna - Why did high A+T content create problems for the Plasmodium falciparum genome project?

The main paper for the Plasmodium palciparum genome project (Gardner et al., 2002) repeatedly mentioned that the unusually high A+T content (~80%) of the genome caused problems. For example they imply that it prevented them using a clone-by-clone approach:

Also, high-quality large insert libraries of (A + T)-rich P. falciparum DNA have never been constructed in Escherichia coli, which ruled out a clone-by-clone sequencing strategy.

And that it made gene annotation difficult:

The origin of many candidate organelle-derived genes could not be conclusively determined, in part due to the problems inherent in analysing genes of very high (A + T) content.

Question:
What is the biological significance of high A+T content, and why would it cause problems in genome sequencing?

Ref:
Gardner, M.J., Hall, N., Fung, E., White, O., Berriman, M., Hyman, R.W., Carlton, J.M., Pain, A., Nelson, K.E., Bowman, S., Paulsen, I.T., James, K., Eisen, J.A., Rutherford, K., et al. (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 419 (6906), 498–511.

evolution - What are the evolutionary niches differentiating an apple from a pear?

I wouldn't be suprised if no one has hard data on this. In any case there is more than one answer to this question.

Niches are defined more than one way.

In the narrowest definition, the niche might be defined as the ability of the different trees to grow in different terrains or be eaten by different animals in the same environment. I can't comment on that, but in most environments, there is in practice little difference between the two. The classic example of this is Darwin's finches - where a single species eventually becomes many, each specializing in a specific kind of lifestyle (what they eat/where they net) on the islands of the Galapagos where they were situated.

In the broadest definition of niche, they do have a different niche because they are different species, and their differences allow them to be eaten by different people. In their highly domesticated environs, apples and pears each have a niche defined by the fact that they are both stocked in the produce sections for people to eat as they choose.

Its worth mentioning that Darwin determined that different organisms can inhabit the same niche. In his case he saw that on different islands of the Galapagos he saw that the finches had evolved into different species, each of which took up the role that a different bird might have back home. so its possible that apples and pears may have substantially the same niche in their respective evolutionary histories.

evolution - How and why did mouth and nasal cavity evolve separate?

Neither the nostrils nor the mouth originally evolved for breathing. Fish have (two pairs of) nostrils which they use to smell and mouths which they use to eat, but they breathe through their gills. Some lobe-finned fishes (the ancestors to tetrapods) evolved a connection between the posterior nostrils and the oral cavity called choanae. A fossil called Kenichthys is a transitional form in this development. The evolutionary reasons behind this development are not particularly well understood. (This should not be surprising when discussing something that happened in the Devonian. It's only recently that the discovery of Kenichthys ended the controversy of whether chonae in tetrapods are homologous to posterior nostrils in fish.)

Basal reptiles have nostrils and mouths and can breathe through both of them, but do not have a separation between the oral and nasal cavities. The next important development is a secondary palate (which separates the nasal and oral cavities). This allows animals to continue breathing while swallowing food. Animals without such a separation must hold their breath while swallowing. This ability is certainly useful in many situations, and unsurprisingly several solutions to this problem have evolved in different lineages.

dna - Why is the 3'UTR region highly methylated in most of the human genes?

According to Choi et al. Genome Biology 2009, 10:R89, DNA methylation at both coding boundaries may regulate transcription elongation and stabilize splicing by reducing the occurrences of exon skipping.

From the abstract:

Here we report a genome-wide observation of distinct peaks of
nucleosomes and methylation at both ends of a protein coding unit.
Elongating polymerases tend to pause near both coding ends immediately
upstream of the epigenetic peaks, causing a significant reduction in
elongation efficiency. Conserved features in underlying protein coding
sequences seem to dictate their evolutionary conservation across
multiple species. The nucleosomal and methylation marks are commonly
associated with high sequence-encoded DNA-bending propensity but
differentially with CpG density. As the gene grows longer, the
epigenetic codes seem to be shifted from variable inner sequences
toward boundary regions, rendering the peaks more prominent in higher
organisms.

Their data (figures 1 and S2), however, do not support a generalized increase in the 3' UTR regions in either human T cells, mouse liver, yeast or flies.

molecular biology - What is the highest competency possible for E coli?

This is a great question as I just made my own "homebrew" chemically competent cells.

There are a vast variety of E. coli strains that are commonly used for cloning. They may be transformed chemically by heat shock method, or electrically by electroporation (a brief summary may be found here). These can be made in the lab manually, or purchased commercially from reputable vendors (I recommend Invitrogen/Life Tech, NEB, Promega).

Chemically competency is achieved using Hanahan's method or some a variation thereof. The method involves washing the bacteria in a series of buffers containing various di-valent cation chloride salts (CaCl₂, RbCl₂, etc). Commercially available E. coli are sold as "regular" and "ultra" competent, ranging from 10⁶ to 10⁶ for regular, and the highest I've seen is 10¹⁰ cfu/µg DNA. For maximum efficiency, especially since a small plasmid such as yours will be easy to transform, electroporation is a more efficient technique, but requires specialized equpment. Electroporation can yield >10¹⁰ cfu/µg. For standard cloning practices, regular or high efficiency chemically competent E. coli are perfectly adequate. Since you mentioned you are creating a library, you certainly want to go with ultra high efficiency chemical transformation or, if you have the necessary equipment, electroporation. I have not seen electroporation efficiencies reported > 5x10¹⁰.

Transformation efficiencies are standardized by reporting a rate as the number of colony forming units (cfu) per µg of some control plasmid, typically pUC19. Transformation efficiency is very sensitive to many factors, including heat shock time, cooling times, thawing time, amount and size of plasmid DNA. All things being equal, the size and quantity of DNA are important for your purposes. Transformation efficiency generally increases with quantity of DNA, but there is a saturation point, and evenetually having too much DNA decreases the yield. Also, efficiency decreases linearly with plasmid size. For example, pUC19, the control vector, is ~2.7 kbp, and your library plasmid is ~6.6 kbp, so you should expect for the same mass of DNA you will have lost 60% transformation due to plasmid size. This loss is due to the physically larger size of DNA being more difficult to move into the bacteria.

botany - Does the use of "var", "x", and/or "ssp" in a scientific name provide specific information?

Let me clarify my answer since it is lower quality than people may like. To answer the question, my friend is a horticulist and has given a more detailed answer.

Subspecies is the most generic, taxonomically-defined term one rank order lower than species. The subspecies (either an individual subspecies, or collective group of subspecies) are defined to be genetically or morphologically distinct among other subspecies belonging to the same species, yet still produce viable offspring from interbreeding. For example, every type of dog is a sub-species of Canis familiaris, and all dogs are capable of breeding with each other. Subspecies, abbreviated Ssp, may refer to an individual subspecies type, or a collective group of related subspecies when a distinct subset is difficult to define or unknown.

There are a variety of terms that are analogous to subspecies, when the strict definition does not apply, or a different term has been used historically. For example, a varietal is the botanical term, and refers to a named subspecies. For example, grapes are a family of species, divided into the various subspecies, and a varietal is a specific subspecies that we refer to by a more common or familiar name, such as Riesling, Chardonnay, etc. (Grapes are a slightly different case in which most grapes are produced from hybrids or crosses for reasons of withstanding environmental conditions avoiding insect prey and fungal infections.) Microbiology has yet another distinction, called a strain. Strains are often genetically different, and yet all belong to a species. For example, several E. coli strains exist. In contrast, viruses are also given strain/subspecies names which don't technically fit the ICZN definition of interbreeding. Cultivar is a similar term, also from the field of botany. Cultivar is specifically applied to plants that are grown for some agricultural benefit, and have been bred or altered by humans for some reason.

In short, there is a zoological definition of subspecies set out in ICZN. This definition requires viable interbreeding among subspecies belonging to the same species. The term applies to individuals, or groups, as the context dictates. Analogous terms of subspecies are applied (adapted?) to other realms of biology, such as botany, microbiology and virology, but the definitions are slightly different, but consistent within the branch of biology (eg, viruses can't breed so they don't fit the ICZN definition, but the term applies). Ssp can also be qualitatively used to describe an unknown subset of a species (I've seen this used in microbiology mostly).

Crosses/hybrids are indicated with the "x" in the nomenclature. Since I found this confusing, I'm copying from Wikipedia.

From a taxonomic perspective, hybrid refers to offspring resulting
from the interbreeding between two animals or plants of different
species.

1. Hybrids between different subspecies within a species
   (such as between the Bengal tiger and Siberian tiger) are known as
   intra-specific hybrids. Hybrids between different species within the
   same genus (such as between lions and tigers) are sometimes known as
   interspecific hybrids or crosses. Hybrids between different genera
   (such as between sheep and goats) are known as intergeneric hybrids.




2. The second type of hybrid consists of crosses between populations,
   breeds or cultivars within a single species. This meaning is often
   used in plant and animal breeding, where hybrids are commonly produced
   and selected because they have desirable characteristics not found or
   inconsistently present in the parent individuals or populations. This
   flow of genetic material between populations or races is often called
   hybridization.

There are some horticultural examples of crosses in which an example of interspecific and intergeneric hybrids, and others, are shown.