Human Genomic YAC inserts on Chromosome 10 Jul 29, 2012 16:24:04 GMT -5
Post by skyship on Jul 29, 2012 16:24:04 GMT -5
Not just one insert within it are others. And it replicates:
"Entrez Gene summary for HK1:
Hexokinases phosphorylate glucose to produce glucose-6-phosphate, the first step in most glucose metabolism pathways.
This gene encodes a ubiquitous form of hexokinase which localizes to the outer membrane of mitochondria. Mutations in
this gene have been associated with hemolytic anemia due to hexokinase deficiency.
Alternative splicing of this gene
results in five transcript variants which encode different isoforms, some of which are tissue-specific. Each isoform
has a distinct N-terminus; the remainder of the protein is identical among all the isoforms. A sixth transcript
variant has been described, but due to the presence of several stop codons, it is not thought to encode a protein.
Note it is in the mitochondrial:
summary for HK1:
Mitochondrial Hexokinases catalyze the first essential step of glucose metabolism, the conversion of glucose
into glucose-6-phosphate. This directly couples extramitochondrial glycolysis to intramitochondrial
oxidative phosphorylation. There are two subtypes of mitochondrial hexokinases, HK1 and HK2, each of which
has four isozymes designated hexokinase I, II, III and IV. HK1 and HK2 are both localized to the outer
mitochondrial membrane, with HK1 being found in nervous tissue and HK2 in insulin-responsive tissues such as
skeletal muscle. Activation of mitochondrial hexokinases is regulated by Akt/PKB-mediated phosphorylation
and they are subject to inhibiton by their end product, glucose-6-phosphate. In addition to glucose
metabolism, mitochondrial hexokinases have been implicated in antiapoptotic and cell survival signaling.
It does not encode a protein because it is artificial/synthetic
so what are all the parts. Non Chromosomal material.
EcoRI and BamHI involved.
First described in 1983 by Murray and Szostak, a YAC is an artificially constructed chromosome and contains the telomeric, centromeric, and replication originsequences named autonomous replicating sequence needed for replication and preservation in yeast cells. A YAC is built using an initial circular plasmid, which is typically broken into two linear molecules using restriction enzymes; DNA ligase is then used to ligate a sequence or gene of interest between the two linear molecules, forming a single large linear piece of DNA..
A typical YAC consists of centromere element (CEN) for chromosome segregation during cell division, telomere and origin of replication (ori) were isolated and joined on plasmid constructed in E.coli. For cloning purpose YAC is digested with restriction enzymes and recombinants are produced by inserting a large fragment of genomic DNA. This molecule can be maintained in yeast as YAC.  The transformant that contain YAC can be identified by red/white color selection. Non-transformed yeast contain white colonies. Red colonies of yeast contain the YAC molecule. Insertion of the DNA molecule into SnaB1 site, inactivates SUP4 its protein will not be expressed. This means colonies containing a YAC molecule with a DNA insert are in fact white.
Red colonies can therefore be excluded as they have no insert, growth on a medium without tryptophan or uracil will exclude colonies with no YAC vector. This is because the YAC vector encodes proteins that facilitate growth in the absence of these essential nutrients, whilst normal yeast are not able to grow in their absence.
INCIDENCE OF ARTIFACTS IS LOW???
"Yeast expression vectors, such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids), have an advantage over bacterial artificial chromosomes (BACs) in that they can be used to express eukaryotic proteins that require posttranslational modification.
However, YACs are significantly less stable than BACs, producing "chimeric effects": artifacts where the sequence of the cloned DNA actually corresponds not to a single genomic region but to multiple regions. Chimerism may be due to either co-ligation of multiple genomic segments into a single YAC, or recombination of two or more YACs transformed in the same host Yeast cell.  The incidence of chimerism may be as high as 50%. . Other artifacts are deletion of segments from a cloned region, and rearrangement of genomic segments (such as inversion). In all these cases, the sequence as determined from the YAC clone is different from the original, natural sequence, leading to inconsistent results and errors in interpretation if the clone's information is relied upon. Due to these issues, the Human Genome Project ultimately abandoned the use of YACs and switched to bacterial artificial chromosomes, where the incidence of these artifacts is very low."
They produce chimeras