Scientists map genome sequence of mustard

Chinese scientists have mapped the genome sequence of allopolyploid Brassica juncea, or mustard, a vegetable commonly used in Chinese cooking.

Zhejiang University's Zhang Mingfang, who is a member of the research program, said Tuesday that the sequencing would help scientists understand and improve the agriculturally important vegetable.

The research paper was published in the Nature Genetics journal.

Brassica juncea, known as "jei cai" in its native China, contains a diverse range of oilseed and vegetable corps important for human nutrition. It mainly grows south of the Yangtze River.

Zhang said under the program, the team has, for the first time, analyzed the cause of mustard's different genetic expressions.

He said mustard used for pickling and oil can bring great economic and social benefits. China has 133,000 hectares of mustard for pickling.

Pickled mustard uses a variant of Brassica juncea. Once processed, the stem retains its crisp texture.

Yang Jinghua, one of the authors of the paper, said scientists had previously published the genome maps of Chinese cabbage and kale. The genome of jei cai has more flexible phenotypes and a complex evolutionary process, which made it harder to decode, he said. The vegetable has double genomes after its natural hybridization between Chinese cabbage and black mustard.

For future application, Yang said they aim to develop a more hardy variant of the plant, which will result in larger yields.

"Some of the Brassica juncea are better at resisting disease, but others are the complete opposite. We can improve it through molecular breeding if we find the genes which determine the strength of disease resistance," he said.

SAN FRANCISCO, Aug. 28 -- Researchers at the University of California, Berkeley, have found a way to boost the efficiency of a gene-editing tool, known as clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9), so that it cuts and disables genes up to fivefold in most types of human cells. While the key to figuring out the role of genes or the proteins they code for in the human body or in disease is disabling the gene to see what happens when it is removed, CRISPR-Cas9 is the go-to technique for knocking out genes in human cell lines to discover what the genes do and holds the promise of accelerating the process of making knockout cell lines. However, researchers must sometimes make and screen many variations of the genetic scissors to find one that works well. In the new study, published in a the journal Nature Communications, the UC Berkeley researchers found that this process can be made more efficient by introducing into the cell, along with the CRISPR-Cas9 protein, short pieces of deoxyribonucleic acid (DNA) that do not match any DNA sequences in the human genome. The short pieces of DNA, called oligonucleotides, seem to interfere with the DNA repair mechanisms in the cell to boost the editing performance of even mediocre CRISPR-Cas9s between 2½ and 5 times. "It turns out that if you do something really simple - just feed cells inexpensive synthetic oligonucleotides that have no homology anywhere in the human genome - the rates of editing go up as much as five times," said lead researcher Jacob Corn, the scientific director of UC Bekeley's Innovative Genomics Initiative and an assistant adjunct professor of molecular and cell biology. The technique boosts the efficiency of all CRISPR-Cas9s, even those that initially failed to work at all. Corn portrays CRISPR-Cas9 gene editing as a competition between cutting and DNA repair: once Cas9 cuts, the cell exactly replaces the cut DNA, which Cas9 cuts again, in an endless cycle of cut and repair until the repair enzymes make a mistake and the gene ends up disfunctional. Perhaps, he said, the oligonucleotides decrease the fidelity of the repair process, or make the cell switch to a more error-prone repair that allows Cas9 to more readily break the gene. The next frontier, he was quoted as saying in a UC Berkeley news release, is trying to take advantage of the peculiarities of DNA repair to improve sequence insertion, in order to replace a defective gene with a normal gene and possibly cure a genetic disease.

LONDON, Aug. 26 -- An international team of researchers has discovered a gene that is linked to the regulations of our coffee consumption, a study says.Previous studies have investigated the biological mechanisms of caffeine metabolism. The new findings suggest that the gene reduces the ability of cells to breakdown caffeine, causing it to stay in the body for longer.

According to the study published Friday by the University of Edinburgh, the team analyzed genetic information from 370 people living in a small village in south Italy and 843 people from six villages in northeast Italy.

They found that people with a DNA variation in a gene called PDSS2 tended to consume fewer cups of coffee than people without the variation. The effect was equivalent to around one fewer cup of coffee per day on average, according to the study.

The researchers carried out the same study in a group of 1,731 people from the Netherlands. The result was similar but the effect of the gene on the number of cups of coffee consumed was slightly lower.

One explanation is that the different styles of coffee that are drunk in the two countries lead to the difference, says the researchers.

In Italy, people tend to drink smaller cups such as espresso while people in the Netherlands prefer larger cups that contain more caffeine overall.

The results of this study add to existing research suggesting that our drive to drink coffee may be embedded in our genes, said Dr Nicola Pirastu, Chancellor's Fellow at the University of Edinburgh's Usher Institute.

Pirastu is one of the authors of the study.

However, larger studies are needed to confirm the discovery and also to clarify the biological link between PDSS2 and coffee consumption, says Pirastu.

The study has been published in the journal Scientific Reports.

Researchers find way to boost CRISPR-Cas9 efficiency

SAN FRANCISCO, Aug. 28 -- Researchers at the University of California, Berkeley, have found a way to boost the efficiency of a gene-editing tool, known as clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9), so that it cuts and disables genes up to fivefold in most types of human cells. While the key to figuring out the role of genes or the proteins they code for in the human body or in disease is disabling the gene to see what happens when it is removed, CRISPR-Cas9 is the go-to technique for knocking out genes in human cell lines to discover what the genes do and holds the promise of accelerating the process of making knockout cell lines. However, researchers must sometimes make and screen many variations of the genetic scissors to find one that works well. In the new study, published in a the journal Nature Communications, the UC Berkeley researchers found that this process can be made more efficient by introducing into the cell, along with the CRISPR-Cas9 protein, short pieces of deoxyribonucleic acid (DNA) that do not match any DNA sequences in the human genome. The short pieces of DNA, called oligonucleotides, seem to interfere with the DNA repair mechanisms in the cell to boost the editing performance of even mediocre CRISPR-Cas9s between 2½ and 5 times. "It turns out that if you do something really simple - just feed cells inexpensive synthetic oligonucleotides that have no homology anywhere in the human genome - the rates of editing go up as much as five times," said lead researcher Jacob Corn, the scientific director of UC Bekeley's Innovative Genomics Initiative and an assistant adjunct professor of molecular and cell biology. The technique boosts the efficiency of all CRISPR-Cas9s, even those that initially failed to work at all. Corn portrays CRISPR-Cas9 gene editing as a competition between cutting and DNA repair: once Cas9 cuts, the cell exactly replaces the cut DNA, which Cas9 cuts again, in an endless cycle of cut and repair until the repair enzymes make a mistake and the gene ends up disfunctional. Perhaps, he said, the oligonucleotides decrease the fidelity of the repair process, or make the cell switch to a more error-prone repair that allows Cas9 to more readily break the gene. The next frontier, he was quoted as saying in a UC Berkeley news release, is trying to take advantage of the peculiarities of DNA repair to improve sequence insertion, in order to replace a defective gene with a normal gene and possibly cure a genetic disease.

Polymerase Chain Reaction (PCR) Analysis

PCR analysis is a technique that allows technicians to create millions of precise DNA replications from a single sample of DNA. In fact, DNA amplification alongside PCR can let forensic scientists perform DNA analysis on samples that are as tiny as only a couple of skin cells. In contrast to some other DNA analysis techniques, PCR analysis has the advantage of analysing minuscule sample sizes, even if they are degraded although they must not be contaminated with DNA from other sources during the collection, storage and transport of the sample.

DNA MICROARRAY PROTOCOL

i)           Set-up the following Pre-Hybridisation solution in a Coplin Jar and           incubate at65°C during the labeling incubation period to equilibrate. 20X SSC 8.75 ml 20% SDS 0.25 ml BSA (100 mg/ml) 5.0 ml H2O to 50.0 ml

ii)            Label control and test genomic DNA as follows:- CONTROL TEST Genomic DNA ˜ 2 mg ˜ 2 mg Random Hexamers (3 mg/ml) 1 ml 1 ml H2O to 41.5 ml to 41.5 ml Heat at 95ºC for 5 minutes. Snap cool on ice and briefly centrifuge. 10X buffer 5 ml 5 ml dNTP's (5mM each dATP, dGTP & dTTP, 2mM dCTP) 1 ml 1 ml Cy-labelled dCTP 1.5 ml (Cy3) 1.5 ml (Cy5) Klenow fragment (10U/ml) 1 ml 1 ml Incubate at 37°C for 90 minutes.

iii)          Incubate the microarray slide(s) in the Pre-Hybridisation solution for 20 minutes at65°C, beginning just before the end of the labelling reactions incubation time at37°C.

iv)          Combine the control and test reactions and purify using the Qiagen MinElute PCR Purification kit, using a two step wash stage using 500 ml then 250 ml volumes of Buffer PE and eluting the labeled cDNA from the MinElute column with 14 ml H2O. The columns retain approximately 1 ml, so the final eluted volume will be 13 ml.

v)           Rinse the pre-hybridised microarray slides in H2O for 1 minute, then in isopropanol for 1 minute. Spin at 1500 rpm for 5 minutes to dry slides. Keep in covered slide box. 1 NICK DORRELL - LAST UPDATE FEBRUARY 2004

vi)          Prepare the Hybridisation solution as follows: - Sample 13 ml H2O 26 ml 20X SSC 12 ml 2% SDS 9 ml Heat at 95ºC for 2 minutes. Allow to cool slowly at room temperature and centrifuge for 30 seconds. Add 2 x 20 ml H2O to the corners of the hybridisation chamber. Place a slide into the chamber. Place a LifterSlip™ glass coverslip (22 mm x 25 mm) over the array section on the slide using tweezers. Pipette the Hybridisation solution onto the slide at the top of the coverslip. Seal the chamber and incubate in a water bath at 65°C overnight.

vii)         Prepare Wash solutions as follows: - Wash A (1X SSC 0.5% SDS) Wash B (0.06X SSC) 20X SSC 20 ml 2.4 ml 20% SDS 1 ml H2O to 400 ml to 800 ml Incubate Wash A solution at 65ºC overnight. Dispense 400 ml volumes into three glass slide washing dishes. Remove slide(s) from the hybridisation chambers and gently remove coverslip(s) by rinsing in Wash A. Place slide(s) in a slide rack and rinse with agitation for 5 minutes. Transfer slide(s) to a clean slide rack and rinse with agitation in Wash B(i) for 2 minutes, then in Wash B (ii) for a further 2 minutes. Spin at 1500 rpm for 5 minutes to dry slide(s).

viii)      Scan slide(s) using Affymetrix 418 scanner and analyse data

（NICK DORRELL - LAST UPDATE FEBRUARY 2004）

Protocol for Whole Animal/ Isolated Organ Vascular Perfusion Fixation( of rats/mice )

Advantages:

• Fixation begins immediately after arrest of systemic circulation.   This  minimizes  the

alteration of cell structure resulting from post-mortem effects.

•  Under in  situ  conditions,  vascular  perfusion  results  in  a  uniform  and  rapid

dissemination of fixative into all parts of the tissue  via the vascular bed, resulting  in an

increased depth and rate of actual fixation.

• The manipulation  of tissues  after the  arrest  of the  systemic  circulation  but  prior  to

fixation is minimized resulting in many fewer artifacts.

• Many organs/ tissues may be effectively fixed at one time, thus maximizing  the use  of

each animal.

• In  the  case  of  immunocytochemical  procedures  employing  relatively mild  fixation

conditions,  fewer  autolytic  artifacts  result;  redistribution  or  translocation  of  cellular

components is minimized; and greater immunocytochemical activity is retained.

Pre-perfusion:

1.  The rat/mouse  is retrieved  from  the  animal  quarters  and  brought  upstairs  to  the

surgery room in a cage. The person  removing  the  animal  should  sign  and  date the

animal inventory card ( yellow card ) in the out column.

2. The  rat/mouse  is  weighed  and  injected  with  a  mixture  consisting  of  ketaset(75

mg/ml) + xylazine(5 mg/ml). The recommended  injectable  dose  of this  anesthetic  is  1

ml/gm of body weight, IM.  Allow 10 to 15 minutes for anesthesia to occur, indicated  by

the  loss  of  sensory/  reflex  response,  i.e.  non-  response  to  tail  pinching,  or  paw

pinching. (Note: Metofane, as  an  inhaled  anesthetic,  may  be  used  instead  of  the

ketaset/ xylazine mixture. In this case, assistance of another person is advised.)

3. Once anesthetized, and during the surgical procedures for whole  animal  or isolated

organ  perfusion,  care  should  be taken to  prevent  heat  loss  in the  anesthetized

animal. They are  quite prone to hypothermia.( use of a lamp is recommended )

4. People should consult Michael for the perfusion method for their particular needs.  In

general, an isolated organ perfusion  will  yield the best results,  but concern  should  be

taken to ensure maximum, most efficient use of the rat/mouse.    For example,  in  the

case of an isolated perfusion of the heart/lungs, the  rest  of the  animal  might  still  be

suitable for biochemistry.5. In most instances, the rat will expire during  the perfusion.  However, in cases  where

the animal survives or lingers, cardiac puncture is the appropriate means of sacrifice.

6.  Upon  completion  of  the  procedure  the  carcass  is  wrapped  in  a  surgical  pad/  or

benchcoat, placed in a plastic bag, and  returned  to the  animal  quarters  in  the  cage.

The carcass is placed in  the freezer; and check that the animal  inventory card ( yellow

card ) is signed off.

Recommended routes for vascular perfusion:

• Whole animal- descending aorta or vena cava.

• Central nervous system/ pituitary- aorta, via the left ventricle.

• Kidney- descending aorta, proximal to its distal bifurcation.

• Liver- portal vein

Perfusion Protocol

Perfusion  pressure,  in  most  instances,  should  be  maintained  between  60  and  100

mm Hg. Use a sphygmomanometer or gravity feed apparatus... we have both!!!

1. Generally, it is optimal to aerate/oxygenate the flush and fixation prior to beginning.

This can be maintained during the perfusion.

2. Flush the animal/organ first with 1XPBS containing  1% sodium  nitrite, pH 7.4 for 30

seconds.

3. Follow up flush with perfusion of fixative for 5 minutes. Fixative contains:

3% formaldehyde (freshly prepared from paraformaldehyde);

1.5% glutaraldehyde

2.5%  sucrose

contained in 100mM cacodylate, pH 7.4

200mls of fixative should be plenty for a mouse; 500 mls for a rat.

4. After  5 minutes  of  continuous  perfusion,  organs  can  be  harvested;  appropriately

dissected  in  fixative/buffer  (100mM  cacodylate,  2.5%  sucrose,  pH  7.4);  and  tissue

pieces allowed to fix an additional 1 hour.

5. Wash in 0.1M Cac/2.5% sucrose pH 7.4 3X 15' EA.6. Post-fix with Palade's OsO4 for one hour on ice, light tight, under hood.

5 ml Palade's 1% OsO4 = 1 ml Acetate-veronal stock

+ 1.25 ml 4% OsO4

+ 1 ml 0.1N HCl

+ 1.75 ml ddH2O

Acetate-veronal stock = 1.15 g NaAcetate Anhydrous

(J.T. Baker 1-3470)

+ 2.943 g NaBarbituate (Veronal)

labelled Barbital--(Sigma B-0500)

to 100 ml with ddH2O

7. Rinse 1X with Kellenberger, then incubate for 1 to 2 hrs. at RT. (or preferably

overnight)

10 ml Kellenberger = 2 ml Acetate Veronal Stock

+ 2.8 ml 0.1N HCl

+ 5.1 ml ddH2O

+ 0.05 g Uranyl Acetate

Check pH with paper before adding UA.

(Should be ~6)

8. One quick rinse in ddH2O, then one quick rinse in 50% ethanol.

9. Dehydrate with graded series of cold (4

0

C) ethanol (70, 95, 100); then three 10’

washes in fresh 100% ETOH at rm. temp; and then finally two 5’ exchanges with

propylene oxide (PO).

14. Place in 50% PO/50% Epon (can be old) overnight, uncovered under vacuum (or

hood).

10. Replace with fresh, 100% epon, and leave under vacuum for 2-6 hours.

11. Embed in fresh, 100% Epon. Put typed or pencil-written label in dummy capsules

with wooden stick, at least two capsules per sample. Pour tissue out of tube into

mincing dish. Place tissue in flat mold with small amount of Epon (to avoid curling)

with a wooden stick and place in 60o oven overnight.

Protocol for Whole Animal/ Isolated Organ Vascular Perfusion Fixation( of rats/mice )

Advantages:

• Fixation begins immediately after arrest of systemic circulation.   This  minimizes  the

alteration of cell structure resulting from post-mortem effects.

•  Under in  situ  conditions,  vascular  perfusion  results  in  a  uniform  and  rapid

dissemination of fixative into all parts of the tissue  via the vascular bed, resulting  in an

increased depth and rate of actual fixation.

• The manipulation  of tissues  after the  arrest  of the  systemic  circulation  but  prior  to

fixation is minimized resulting in many fewer artifacts.

• Many organs/ tissues may be effectively fixed at one time, thus maximizing  the use  of

each animal.

• In  the  case  of  immunocytochemical  procedures  employing  relatively mild  fixation

conditions,  fewer  autolytic  artifacts  result;  redistribution  or  translocation  of  cellular

components is minimized; and greater immunocytochemical activity is retained.

Pre-perfusion:

1.  The rat/mouse  is retrieved  from  the  animal  quarters  and  brought  upstairs  to  the

surgery room in a cage. The person  removing  the  animal  should  sign  and  date the

animal inventory card ( yellow card ) in the out column.

2. The  rat/mouse  is  weighed  and  injected  with  a  mixture  consisting  of  ketaset(75

mg/ml) + xylazine(5 mg/ml). The recommended  injectable  dose  of this  anesthetic  is  1

ml/gm of body weight, IM.  Allow 10 to 15 minutes for anesthesia to occur, indicated  by

the  loss  of  sensory/  reflex  response,  i.e.  non-  response  to  tail  pinching,  or  paw

pinching. (Note: Metofane, as  an  inhaled  anesthetic,  may  be  used  instead  of  the

ketaset/ xylazine mixture. In this case, assistance of another person is advised.)

3. Once anesthetized, and during the surgical procedures for whole  animal  or isolated

organ  perfusion,  care  should  be taken to  prevent  heat  loss  in the  anesthetized

animal. They are  quite prone to hypothermia.( use of a lamp is recommended )

4. People should consult Michael for the perfusion method for their particular needs.  In

general, an isolated organ perfusion  will  yield the best results,  but concern  should  be

taken to ensure maximum, most efficient use of the rat/mouse.    For example,  in  the

case of an isolated perfusion of the heart/lungs, the  rest  of the  animal  might  still  be

suitable for biochemistry.5. In most instances, the rat will expire during  the perfusion.  However, in cases  where

the animal survives or lingers, cardiac puncture is the appropriate means of sacrifice.

6.  Upon  completion  of  the  procedure  the  carcass  is  wrapped  in  a  surgical  pad/  or

benchcoat, placed in a plastic bag, and  returned  to the  animal  quarters  in  the  cage.

The carcass is placed in  the freezer; and check that the animal  inventory card ( yellow

card ) is signed off.

Recommended routes for vascular perfusion:

• Whole animal- descending aorta or vena cava.

• Central nervous system/ pituitary- aorta, via the left ventricle.

• Kidney- descending aorta, proximal to its distal bifurcation.

• Liver- portal vein

Perfusion Protocol

Perfusion  pressure,  in  most  instances,  should  be  maintained  between  60  and  100

mm Hg. Use a sphygmomanometer or gravity feed apparatus... we have both!!!

1. Generally, it is optimal to aerate/oxygenate the flush and fixation prior to beginning.

This can be maintained during the perfusion.

2. Flush the animal/organ first with 1XPBS containing  1% sodium  nitrite, pH 7.4 for 30

seconds.

3. Follow up flush with perfusion of fixative for 5 minutes. Fixative contains:

3% formaldehyde (freshly prepared from paraformaldehyde);

1.5% glutaraldehyde

2.5%  sucrose

contained in 100mM cacodylate, pH 7.4

200mls of fixative should be plenty for a mouse; 500 mls for a rat.

4. After  5 minutes  of  continuous  perfusion,  organs  can  be  harvested;  appropriately

dissected  in  fixative/buffer  (100mM  cacodylate,  2.5%  sucrose,  pH  7.4);  and  tissue

pieces allowed to fix an additional 1 hour.

5. Wash in 0.1M Cac/2.5% sucrose pH 7.4 3X 15' EA.6. Post-fix with Palade's OsO4 for one hour on ice, light tight, under hood.

5 ml Palade's 1% OsO4 = 1 ml Acetate-veronal stock

+ 1.25 ml 4% OsO4

+ 1 ml 0.1N HCl

+ 1.75 ml ddH2O

Acetate-veronal stock = 1.15 g NaAcetate Anhydrous

(J.T. Baker 1-3470)

+ 2.943 g NaBarbituate (Veronal)

labelled Barbital--(Sigma B-0500)

to 100 ml with ddH2O

7. Rinse 1X with Kellenberger, then incubate for 1 to 2 hrs. at RT. (or preferably

overnight)

10 ml Kellenberger = 2 ml Acetate Veronal Stock

+ 2.8 ml 0.1N HCl

+ 5.1 ml ddH2O

+ 0.05 g Uranyl Acetate

Check pH with paper before adding UA.

(Should be ~6)

8. One quick rinse in ddH2O, then one quick rinse in 50% ethanol.

9. Dehydrate with graded series of cold (4

0

C) ethanol (70, 95, 100); then three 10’

washes in fresh 100% ETOH at rm. temp; and then finally two 5’ exchanges with

propylene oxide (PO).

14. Place in 50% PO/50% Epon (can be old) overnight, uncovered under vacuum (or

hood).

10. Replace with fresh, 100% epon, and leave under vacuum for 2-6 hours.

11. Embed in fresh, 100% Epon. Put typed or pencil-written label in dummy capsules

with wooden stick, at least two capsules per sample. Pour tissue out of tube into

mincing dish. Place tissue in flat mold with small amount of Epon (to avoid curling)

with a wooden stick and place in 60o oven overnight.