Lab Notebook
July 24, 2012
We dissected oysters and seastars. Pictures are below. The water vascular system in the sea star was really fascinating, but the sea star was very hard to dissect.
July 25, 2012 Case study: RLO in Arminia californica
Accession Number: 12-1-4
Gross examination: Individual has 7 lesions on the dorsal surface, possible inflammation in cerrata
Mass: 3.2 g
Length: 36.7 mm
For PCR and histology we took two samples because we were not sure which was the digestive gland:
12-1-4A: whitish tissue
12-1-4B: orange tissue
Accession Number: 12-1-7
Gross examination: individual has 17-19 lesions
Mass: 8.6g
Length: 57.7mm
1 sample for histology, one for PCR (stored in EtOH)
DNA Extraction
Accesion number 12-1-4B
Mass: 0.1 g
Extraction according to lab exercise protocol.
PCR
PCR for Universal primers (detecting bacteria), Ehrlichia primers, and WS-RLO primers. The first two used the generic temperature settings for PCR, while the last used an optimized protocol.
Gel electrophoresis
Gel electrophoresis was run for DNA 12-1-4A (extracted by Jamie) and 12-1-4B for all three primers, additional negative and positive controls were added. DNA was detected in the positive controls for the Universal and WS-RLO primers. Not sure what went wrong with the Ehrlichia primers- the positive control should have been positive there as well.
July 29, 2012: Labyrinthulid-eel grass collection and histology
We took a field trip to False Bay and sampled 3 parallel 10m transects in the shallow end- the tide was coming in too quickly to sample eelgrass in deeper water. Along each transect we measured shoot density at 5 locations (2 m apart) in a 0.5 m2 quadrat. In addition, for shoots that were within 6 inches of either side of the transect, we counted the number of lesions on the longest blade for each shoot and measured those lesions. We also (in most cases) recorded if the plant was flowering or not. We collected blades with lesions to bring back to the lab. We also measured water quality parameters (pH, temp, salinity, turbidity).
**Next trip, I would be interesting in measuring the diversity of grazers and epiphytes on the eel grass and comparing diversity within and among transects.
Back at the lab
Culturing Laby: We identified healthy and unhealthy blades of eelgrass to use for culturing laby. Unhealthy blades had lesions with a black outline. To culture the laby, we wiped epiphytes off of the eelgrass, photographed it, and then dipped a small section in ethanol to kill the outer surface. We put the blades onto agar petri-dishes that contained agar, BSA, pen-strep (antibiotics) and a food source (not sure what this is).
Histology of laby:
We examined histological slides of eelgrass from samples previously collected by Catherine. Laby has a spindle shape and stains dark purple (I hope to add a picture soon!). It is often surrounded by mucous (used for transportation of laby as well as a protective coat). Identification of laby was challenging. We found many spindle-shaped structures in 'healthy' slides, but they often lacked mucous and did not stain darkly. Drew has sent off a few pictures to Lisa Muehler (sp?) who has worked with this patho-system and can hopefully clarify our questions. Tomorrow we will do PCRs on samples collected by Catherine (the same samples that we looked at the histology for).
PCR of laby:
Today we divided up samples of laby for DNA extraction and PCR. The extractions went really smoothly. Today we set up the thermocycler. Everything went really smoothly. For both extraction and the PCR, our methods did not differ at all from the protocol. My sample is Accession # PC D8.
Prep of laby for innoculations:
We were interested to see if we could use small pieces of laby for innoculation experiments. Would the plants die if we cut them? Are there any other issues to consider? To test this, I have removed 5 clean blades of laby from Sandy's tanks and cut them into 2 cm sections. They are floating in sea water in the lab. We can check their health in a few days and by Monday, we should have enough laby in broth (thanks Lisa for making this!) to try innoculations.
My Favorite Gene
Maya Groner
Corticotropin-releasing hormone
Corticotropin-releasing hormone (CRH), originally named corticotropin-releasing factor (CRF), and also called corticoliberin, is a__peptide____hormone__ and__neurotransmitter__ involved in the__stress response__.
Here is CRH interacting with an agonist (from H. sapiens)
More info:
__http://en.wikipedia.org/wiki/Corticotropin-releasing_hormone__
This is from amiGO. It shows all of the actions of this gene (or something close to that)
Taxa of interest
Rana pipiens
Rana pipiens
Taxonomy ID: 8404Genbank common name: northern leopard frog
Inherited blast name: frogs & toads
Rank: species
Genetic code:__Translation table 1 (Standard)__
Mitochondrial genetic code:__Translation table 2 (Vertebrate Mitochondrial)__
Other names:
| synonym: |
Lithobates pipiens |
| synonym: |
Pantherana pipiens |
__Lineage__( full )
__cellular organisms__;__Eukaryota__;__Opisthokonta__;__Metazoa__;__Eumetazoa__;__Bilateria__;__Coelomata__;__Deuterostomia__;__Chordata__;__Craniata__;__Vertebrata__;__Gnathostomata__;__Teleostomi__;__Euteleostomi__;__Sarcopterygii__;__Tetrapoda__;__Amphibia__;__Batrachia__;__Anura__;__Neobatrachia__;__Ranoidea__;__Ranidae__;__Rana__;__Pantherana__
134 AA sequence
10 20 30 40 50 60
SSKSSNGAPA YHPFLLRMGE EYFLRLGNLH KSPLGGSRVG DVSSSNFVRA VQQLQPQQWV
70 80 90 100 110 120
GQQELRAGSM VDGADSSPFS AQEEPTERGK RSEEPPISLD LTFHLLREVL EMARAEQIAQ
130
QAHSNRKLMD IIGK
Transcriptome
>ENA|ABG91818|ABG91818.1 Rana pipiens (northern leopard frog) partial corticotropin-releasing hormone : Location:1..402
AGCAGCAAATCTTCTAATGGGGCTCCCGCATACCACCCCTTTCTACTCCGTATGGGAGAA
GAGTACTTCCTCCGCCTGGGGAACCTGCACAAGTCCCCCCTGGGAGGCAGCCGAGTCGGG
GACGTCTCGTCCAGTAACTTTGTGCGGGCGGTGCAGCAGCTCCAACCTCAGCAGTGGGTC
GGCCAGCAGGAGCTCCGAGCCGGCAGCATGGTGGACGGAGCCGACTCCTCTCCATTCAGC
GCTCAGGAGGAGCCCACGGAAAGAGGAAAACGTTCGGAGGAGCCCCCCATTTCCCTGGAT
CTCACTTTCCATCTGCTCCGGGAAGTCTTGGAAATGGCCAGAGCCGAGCAGATAGCCCAG
CAAGCCCACAGCAATAGGAAACTGATGGACATCATTGGGAAA
This is a highly conserved sequence: Most hits have E< 10^-10:
MFG-2
Brevinin
Frog antimicrobial peptide; This family consists of the major classes of antimicrobial peptides secreted from the skin of frogs that protect the frogs against invading microbes. They are typically between 10-50 amino acids long and are derived from proteolytic cleavage of larger precursors. Major classes of peptides such esculentin, gaegurin, brevinin, rugosin and ranatuerin are included in this family.
Lots of hits
Sequence:
ORIGIN
1 caataaggtg gtgaatgatt tttcaaaatt tccatataag taaattcctc ccagtgtatg
61 ggcagcttta gctgtgtcca cttactgatg ggaaagctct atgccatagt atgaaacatc
121 agaatgttcc cgctggttat tcaataagag attgtggcat cccgctgtgc cgtattcctt
181 taccattcct ttaccatctt actgtggtca tagaatagta gacaacgtgg cctaaaaata
241 cacatttgtg gccccagtta aatacggtac agtttttgtg taataatcaa tgttgtttta
301 tttaccgata actgtgtctt ctcccaggag aaatgcagag gaagaaagaa gagatgggcc
361 agatggaaag gaagaaacag gtataccact tcttccagga ttggccgcta acctctgtcg
421 gccaatatat tgtacaataa ccaaaaattg ttgaaa
translation="NAEEERRDGPDGKEETGIPLLPGLAANLCRPIYCTITKNC"
Lots of very similar sequences in ranids!
There is a brevinin ‘superfamily’
10 20 30 40 50
....*....|....*....|....*....|....*....|....*....|
__gi 147744594__2 NFLKKSLFLVLFLGFVSISFCDEEKRQDDDEgNEREEKKEIQEdgnQEER51
__gi 13959331__ 2 AFLKKSLFLVLFLAIVPLSICEEEKREEE---NEEKQEDDDQS---EK-R44
__gi 13959332__ 2 AFLKKSLFLVLLLGLISLSICEEEKRENE---VEEEQEDDEQS---ELRR45
__gi 1706455__ 2 DILKKSLFLVLFLGLVSLSICEEEKRENE---DEEKQD-DEQS---EMKR44
__gi 29336871__ 2 LTLKKSMLLLFFLGLVSVSLADDKREDEAEE-GEDKRAAEEER---NVEK47
__gi 29336801__ 2 FTLNKFLLLLFFLGTINLSFCEEENAEEE---RIDEPD-ETDV---EVEK44
__gi 2493352__ 2 FTLKKSLLLLFFLGTINLSLCEEERNAEEE--RRDEPD-ERDV---QVEK45
__gi 29336799__ 2 FTLKKSLLLLFFLGTISLSLC--QREADD---DQ----GEVQQ---EVKR39
__gi 2506236__ 2 FTMKKSLLFLFFLGTISLSLCEEERSADEDD-GG---E-MTEE---EVKR43
__gi 29336553__ 2 FTLKKSLLLLFFLGTISSSLCEQERDSDDED-QGEVTEQVVKR---LV-R46