Spinal Cord Transcriptome Analysis using Suppression Subtractive
Hybridization and Mirror Orientation Selection
Lathia, Kanan B., Yan, Zhi, Clapshaw,
Patric A.1
Solomon
Park Research Institute,
Summary
Comparison of cDNA libraries derived from the spinal cord with those derived from the visual cortex by means of subtractive hybridization resulted in 60 differentially expressed genes.
Key Words: spinal cord transcriptome, suppression subtractive hybridization, mirror orientation selection, Proteolipid protein, ferritin, motor neuron disease, amyotrophic lateral sclerosis
1 Author to whom inquiries concerning this article should be made. Email pclapshaw@solomon.org
Introduction
The spinal cord is important in several areas of medical research. This structure is the site of frequent injuries causing loss of function or paralysis as well as an important site of motor neuron diseases (MNDs), such as amyotrophic lateral sclerosis (ALS) among others. Despite these considerations, little is known of the molecular differences between the spinal cord and other neural structures. Without this knowledge, many of the functions responsible for the onset of these conditions or the lack of regeneration to injury or disease may be overlooked.
A number of studies have compared different regions in the mammalian spinal cord and brain using subtractive hybridization (Kobayashi et al. 1991; Usui et al 1994; Akopian & Wood 1995) serial analysis of gene expression (de Chaldée et al. 2003) and microarray technology (Sandberg et al. 2000). The results from these studies are remarkable in that the number of expressed genes that differ between structures or even between isolated neurons within a particular structure (Tietjen et al. 2003) are small. Given the observation that the cytological architecture of the mammalian nervous system is highly variable and complex, as well as previous estimates that the number of genes specific to the nervous system could exceed half of all mRNA species (Milner & Sutcliffe, 1983) these findings are surprising.
A better approach to the question of genetic expression in nervous tissue would be to compare regions that differ in a known structure or function as has been reported, for example, for opossum spinal cord areas that are able or unable to regenerate (Mladinic et al. 2005). In the present study, we have used the spinal cord, without sensory ganglia to represent motor functions as tester and visual cortex which represents sensory functions as driver. We have separated mRNA from these two regions and have subtracted out the common sequences using suppression subtractive hybridization (SSH) (Diatchenko et al. 1996). This procedure has the advantage of performing normalization and subtraction in a single step allowing the amplification of genes represented by low copy numbers. SSH also allows the identification of unknown genes which would not be detected in microarray analyses.
Although the SSH method is a powerful procedure for comparing cDNA sequences generated from differing sources, it is thought to be susceptible to cDNA sequences remaining after the subtraction process giving rise to false positives. Mirror orientation selection (MOS) is argued to decrease the presence of highly abundant sequences (Rebrikov et al. 2000), therefore, to rule out these background sequences as the source of any differentially expressed genes in these preparations, we have included MOS for comparative analysis with SSH in two of the four subtractions reported here.
Materials and Methods
Overall Approach
Sequences from four separate cDNA libraries from murine spinal cord mRNA subtracted against visual cortex cDNA were analyzed. Initial subtractions were performed using SSH followed by a second round in two of these libraries using MOS. The first two subtractions used the posterior one third of the cerebral cortex as driver and the third and forth subtractions used a smaller amount of underlying white matter with the visual cortical area as driver. The second subtraction was performed with excess cDNA representing Plp exon 7 during both the first and second hybridization steps (see below). Subtractions 1 and 2 were performed on Swiss Webster mice and subtractions 3 and 4 were performed on C57Bl6J mice. The subtractions and subsequent mirror orientation selection analyses are designated by number followed by either SSH or MOS. These subtractions are summarized in Table I. The subtractions will be subsequently designated as shown in Table I as 1-SSH for subtraction 1 using SSH, 3-MOS as subtraction 3 SSH followed by MOS, etc.
Tissue Preparation
Tissues for subtractions 1-SSH and 2-SSH were from 90-110 day old male Swiss Webster mice from Pel Freeze and for subtractions 3-SSH and 4-SSH and the subsequent 3-MOS and 4-MOS from 90-110 day old male C57Bl6J mice housed at the Jackson Laboratories in Bar Harbor, Maine. The animals were sacrificed by carbon dioxide asphyxiation in subtractions 1-SSH and 2-SSH and by cervical dislocation in subtractions 3-SSH and 4-SSH and in all cases; the brains immediately removed and stripped of meninges. Spinal cord central cores were dissected out and visual cortices were manually dissected away from the remainder of the brain. All dissected tissues were frozen on blocks of dry ice. Tissues were shipped to Solomon Park on dry ice and held at -70○C until ready for use.
RNA Isolation
Total RNA was isolated from spinal cord or visual cortex preparations that had been frozen under liquid N2 and powdered in a pre-cooled mortar and pestle before extraction by the Acid:Phenol:Guanidinium (Trizol) method (Chomczynski & Sacchi, 1987). Poly (A)+ RNA was prepared by separation on a MicroPoly(A) PuristTM Kit from Ambion. RNA was quantified by reading the absorbance at 260/280nm and checked for integrity by RNA agarose gel electrophoresis.
Suppression Subtractive Hybridization
Suppression subtractive hybridization was performed using The BD Clontech PCR-Select™ cDNA Subtraction Kit with cDNA from spinal cord as tester and cDNA from visual cortex as driver. Poly (A)+ specimens were prepared as described above and 2 µg from each preparation were used to prepare ds cDNA which was subsequently digested with the endonuclease RSA I. Tester RNA preparations were then divided into 2 subpopulations and each ligated with either adaptor NP1 or NP2R from Clontech. Subtraction and normalization were accomplished in a single step with a 50 fold excess of driver cDNA over tester followed by two rounds of PCR amplification.
Subtraction 2-SSH was accomplished with 100 ng PCR isolated product representing Plp1 exon 7 (primer sequences used were 5’ CCCAGATTTCAGGCTTATCC-3’ and 5’AGCACTTTGGGGAATGACAG-3’) added to both the primary and secondary hybridization steps during the subtraction.
All secondary PCR products were ligated with pCR® 4-TOPO (InVitrogen) and used to transform TOP10 E. coli cells (InVitrogen).
Subtraction Efficiency
Subtraction efficiency was measured by the intensity of the PCR products on agarose gel representing glyceraldehyde-3-phosphate dehydrogenase (G3PDH) before and after the SSH procedures.
Mirror Orientation Selection
Mirror orientation selection was performed on two of the four subtractions as described by (Rebrikov 2003). Ten independent primary PCR tubes were aliquoted from the second hybridization samples from the SSH procedure described above. These aliquots were diluted 1000 fold and 10 cycles of PCR was performed using NP1 and NP2R as primers. The samples were phenol/chloroform/ethanol precipitated and the purified products were digested with the endonuclease XMA I following which the samples were hybridized for three hours at 68○C. The preparation was finally amplified by 19 cycles of PCR using NP2Rs primer only and the products ligated into TOPO 10 vector (InVitrogen) and subsequently used to transform TOP10 E. coli (InVitrogen).
Probe preparation
DNA to be labeled was diluted to a concentration of 2.5-25 ng in 45 µl of TE buffer (10mM Tris HCl Ph 8.0, 1mM EDTA). The sample was denatured for 5 min at 95-100○C then snap cooled on ice for 5 minutes and briefly centrifuged. The denatured DNA was added to the reaction tube (Amersham) containing 50 µCi [32P] dCTP (Perkin Elmer) The resultant mixture was incubated at 37○C for 10 min. The reaction was stopped by adding 5 µl of 0.2M EDTA. Labeled probes were purified using BD Chroma Spin Columns from BD Biosciences.
Differential Screening
Differential screening was performed using the PCR-Select Differential Screening Kit from BD Biosciences. Subtracted clones were sampled (500 clones randomly selected per subtraction) and spotted onto four nylon transfer membranes (Schleicher and Schuell). The membranes were subsequently interrogated with [32P] labeled cDNA (RediprimeII random prime labeling system from Amersham Biosciences) from the forward and reverse subtracted and unsubtracted source materials . Clones exhibiting at least two fold differences only in the forward subtraction were selected for sequencing.
Northern Hybridization Procedure
Total RNA (5-10 µg) from murine spinal cord, visual cortex and kidney was separated by gel electrophoresis on 1% agarose – formaldehyde gel using the NorthernMax Kit (Ambion) and subsequently blotted onto a nylon hybridization membrane (PerkinElmer). Probes containing 1 x 106 cpm/ml [32P] CTP were incubated in hybridization buffer (NorthernMax) at 42○C overnight followed by two15 minute washes with 2X SSC, 0.1% SDS at room temperature and 0.1X SSC, 0.1% SDS twice at 42˚C. The resulting blots were exposed to X-ray film (Kodak) for detection.
Miniprep
The QIAprep miniprep kit from Qiagen was used for plasmid minipreps. Cultures of 1-5ml of E.coli were grown overnight in LB medium and were lysed under alkaline conditions. The lysate was subsequently neutralized and adjusted to high-salt binding conditions. The DNA was purified on a silica-gel membrane.
Sequencing
Sequences from the minipreps
described above were obtained from Davis Sequencing in
Results
Overall Analysis of Differentially Expressed Sequences
From approximately 10,000 colonies across four separate subtractions, 2,000 (500 per subtraction) were randomly selected for analysis. One hundred forty two clones from these colonies were identified by dot blot analysis as differentially expressed in spinal cord and subsequently sequenced and identified using the BLAST programming available through the NCBI. From these sequences, 60 unique genes were identified. The results of these subtractions and the subsequent MOS procedure are summarized in table II
Subtraction Efficiency
Subtraction efficiencies were in excess of 1000 fold. The comparison using PCR identifying G3PDH cDNA representing subtraction 2-SSH is shown in figure 1.
Comparison of SSH and MOS
There was an approximately 50 % reduction in repeated sequences between SSH and MOS in our subtractions as can be seen in Table III. Sequences representing proteolipid protein, for example, are represented by 12 separate colonies in SSH preparations and in 9 when isolations were performed with MOS. The overall representation of repeated sequences were reduced from 38 in SSH libraries to 22 in MOS libraries which makes this procedure marginally useful in these complex subtractions.
Northern Analysis of Myelin Associated Sequences
Two probes representing commonly accepted myelin markers: proteolipid protein and myelin basic protein and two other sequences associated with myelin: myelin associated glycoprotein and ferritin heavy chain were used to verify the differential expression of myelin in the spinal cord versus visual cortex preparations by Northern analyses and are shown in figure 2. As can be seen, the increase in these myelin markers is substantial and is in excess of 10 fold for all of these markers. The Northern blots representing these myelin associated sequences were normalized with beta actin (results not shown).
Northern Analysis of Other
Sequences
Clones represented only once in any of the four subtractions or subsequent MOS selections represented 70 % of the clones examined (42 of the total of 60 unique clones). In order to verify that these sequences were differentially expressed between spinal cord and visual cortex, we randomly selected 20 clones to analyze by Northern analysis. All sequences tested were up-regulated in spinal cord when compared to visual cortex. The results of four analyses are presented in figure 3. All northern analyses were normalized against beta actin (results not shown).
Discussion
General Considerations
Most of the differentially expressed sequences observed (53 out of 61 or 89 %) code for previously observed proteins of known and unknown function while the remaining sequences (7 out of 61 or 11 %) are of hypothetical proteins of unknown function. Only one sequence was located in an intergenic region of the mouse genome.
Nearly half of all sequences observed (44 %) can be assigned to glial cells while approximately one quarter (24 %) are of neural origin. The remaining sequences cannot be assigned to any particular cell type, however, it might be assumed that they are either common to both neural and glial elements or minimally reflect the neuron to glial cell ratio of the known sequences. Approximately 5 % of the clones identified as differentially expressed between the spinal cord and the visual cortex are of mitochondrial origin.
The types of molecules differentially expressed, as with other studies in the nervous system, represent a broad range of products including cell cycle and differentiation proteins; enzymes; heat shock, motor, structural, membrane and transport proteins as well as proteins that are both hypothetical and previously observed with unknown functions. The use of differentially expressed genes will involve an analysis of the patterns of genes as well as a search for any unique products that may be used to distinguish cells or cell populations.
The sequence representing Plp1 accounts for half of all glial sequences and nearly 25 % of all observed sequences. Ferritin heavy chain (FTH), a protein known to be produced in oligodendrocytes in the nervous system, accounts for nearly 20 % of glial cell sequences and nearly 10 % of all observed clones.
Subtraction Efficiency
The preponderance of sequences representing myelin associated cells raises the possibility that SSH and MOS are ineffective in demonstrating differences between complex neural structures such as spinal cord and visual cortex. We feel that this is not the case for several reasons. First, the efficiency of our subtractions as measured by successive PCR rounds of cDNA using primers for G3PDH is in excess of 1000 fold. Second, all subtracted cDNA sequences contained the predicted adaptors both for SSH (NP1 and NP2R) and MOS (NP2Rs) (data not shown). Third, the MOS results are essentially the same as those for SSH, indicating that the initial subtractions were successful. Fourth, the subtractions are highly reproducible. Fifth, the addition of excess Plp1 sequences to the second subtraction (2-SSH) eliminated Plp1 from the sequences exhibited in this subtraction. In this last case, it would be expected that the addition of a higher number of sequences would increase the number of clones exhibited if the effect were due to background. Finally, all Northern analyses surveyed show increased probe activity of all positive clones in the spinal cord compared to visual cortex.
These arguments lead us to conclude that the genes represented by the clones isolated in our procedures are, in fact, differentially expressed between adult, murine spinal cord and visual cortex. Whether this difference is the result of an increase in expression in the spinal cord or a decrease in expression in the visual cortex for any particular sequence, however, is not easily resolved; although the almost complete absence of bands representing many of the sequences in visual cortex would argue that most sequences are truly up-regulated in spinal cord. It is also not clear how extensively the differences between these structures are represented. The absence of the motor neuron marker, Islet-1 would indicate that the sequences do not represent all of the possible differences. The presence of sequences for the myelin markers, CNPase and GFAP as well as many other well known myelin protein sequences does indicate that this tissue is well represented in these subtractions.
The number of sequences in our study is similar to that of previous studies. The fact that when analyzed extensively, most of these sequences represent the more numerous glial elements that form the overwhelming majority of cells in the nervous system raises the possibility that the relatively small number of unique sequences observed in this and other studies is a result of the variation in composition or in turnover rates of myelin in the different brain areas under study. Procedures such as laser capture allowing the isolation of small numbers of specific cell types or methods designed to subtract out myelin elements prior to comparing cDNA from different brain areas could alleviate this obstacle to identifying low copy genes uniquely expressed in specific neurons.
Novel Genes Up-Regulated in Spinal Cord
Seven genes with putative or hypothetical proteins are up-regulated in spinal cord when compared to visual cortex: cerebellar granule cell antiserum positive 14 (Gcap14 or 2900054P12Rik ); medulla oblongata derived AI841971; whole brain derived 2610024G14Rik; clone 6330420E11; 2810437LI3Rik; 2500002L4Rik and A930004K21Rik were identified. These genes and their predicted protein products all represent potentially important differences for cells specific to the spinal cord. Two of these hypothetical products, 2500002L4Rik and A930004K21Rik may represent transcription and translation control and as such present important possibilities for distinguishing cells in this structure from other neural cells. It should be obvious, however, that simply because the product of a gene is not known there is no reason to assume that it has a more important role in distinguishing cells or structures than that of known genes.
Significance of Myelin
The up-regulation of numerous genes associated with myelin in adult spinal cord when subtracted against visual cortex is unexpected. The driver cDNA, in this case the visual cortex with underlying white matter, should present sufficient cDNA from myelin at the observed subtraction efficiencies to eliminate any cDNA from the spinal cord. Additionally, at the age that the animals were tested, the developmental period normally associated with the formation of myelin has been long since completed.
In an exhaustive study quantifying the abundance of regional variations in adult human brain and spinal cord of the myelin proteins; proteolipid protein (PLP), myelin basic protein (MBP) and 2’, 3’ cyclic nucleotide 3’ phosphodiesterase (CNP); spinal cord did not display an excessive amount of PLP when compared to occipital cortex (Trotter et al. 1984). This study also found similar results for CNP, however, MBP was elevated in spinal cord white matter when compared to other structures. These measurements do not coincide with our observations with cDNA sequences for these proteins.
The present study leaves little doubt, however, that many myelin proteins and in particular Plp1 are highly up-regulated in the adult, murine spinal cord when this structure is compared to the sensory dominated visual cortex. Not only is Plp1 represented in every subtraction (except where purposely cancelled by an excess amount of reverse complement Plp1-cDNA during the hybridization procedures) but it was the only gene that was represented by several different exons (results not shown) which indicates that this gene was in very high abundance. All other sequences were represented by single sequences, often in the 3’ untranslated region as would be expected by the method of selection used in the present study.
Two genes products associated with the general function of iron transport are differentially expressed in spinal cord that are known to be produced in the nervous system in glial cells, ferritin heavy chain (FTH) and transferrin (TRF). Both of these proteins bind and sequester iron where present (Bloch et al. 1985), which would make them important in reducing the formation of free radicals in the tissues where they are present.
Our results indicate that the amount of myelin in the spinal cord versus the visual cortex is substantially greater than previously measured or that the turnover of myelin in this structure is greater in the adult murine nervous system. It is also possible that the composition of myelin itself is variable from one area of the nervous system to another as has been shown for astrocytes under various dopamine exposures (Jun et al. 2001). At the very least, it indicates that the influence of myelin in spinal cord should not be underestimated.
Association with disease states
The spinal cord and in particular the motor neurons in this structure are prone to a number of disease states, predominant among which is MND also known as ALS. It is tempting to speculate on a relationship between the current results and any possible cause or causes of this disease.
There is no compelling reason to speculate that a single gene or gene product is the cause of ALS. Indeed, it is hard to imagine that this is the case except for the small percentage of hereditary ALS cases related to the Sod1 mutations described below. Other candidate proteins, for example the hypoxia response element (HRE) in the promoter for vascular endothelial growth factor (VEGF) though initially viewed as a strong candidate (Oosthuye et al. 2001) has failed to yield clear genetic differences in population studies (Lambrechts et al. 2003).
Similarly, there is no a priori reason to assume that any particular cell type or population of cells within an organ is necessarily the source of pathology for the cells that are affected within that structure. In the hereditary form of ALS that has been correlated with mutations in the copper zinc superoxide dismutase (Sod1) gene (Rosen et al. 1993), for example, the mutations that are believed to cause the motor neuron degeneration are expressed ubiquitously in the individuals affected. Why motor neurons preferentially degenerate as a result of these mutations is unknown, however, other intra and extra cellular factors cannot be ruled out. Other mutations, such as alterations in the expression patterns of metabotropic glutamate receptors (mGluRs) groups I-III associated with glial cells have been correlated with ALS (Anneser et al. 2004). Again, why these general myelin alterations appear only to affect motor neurons is difficult to understand.
Despite these caveats, several of the genetic differences observed in the current study deserve attention.
Proteolipid protein, which is primarily associated with myelin in the CNS, is known to act in a paracrine manner to modulate the survival of neurons in tissue culture (Boucher et al. 2004) as well as increase neurodegeneration in mice when under expressed (Griffiths et al. 1998) and late-onset neurodegeneration in mice when over expressed (Anderson et al. 1998). A number of studies have demonstrated that dysregulation of the Plp1 gene affects neuronal function (Boison & Stoffel, 1994, Klugmann et al. 1997). Some evidence also exists that isoforms of this protein are also present in motor neurons (Werner et al. 2001).
N-Myc down regulated gene 4 (Ndrg4) is highly expressed in brain and heart (Zhou et al. 2001), and has been associated with neurite outgrowth (Ohki et al. 2002)
The high incidence of the iron binding proteins ferritin and transferrin in the spinal cord should also not be ignored. As stated earlier, these proteins are noteworthy in their ability to act as a sink for iron which if high would tend to increase the amount of free radical damage to surrounding tissues.
Conclusion
The spinal cord exhibits a number of up-regulated genes when compared to visual cortex, most of which are associated with myelin. These genes are unlikely to be simply the result of background sequences escaping the subtraction process. Several of the up-regulated genes are known to be associated either directly or with conditions that could increase neuronal cell death.
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Classification
of Subtractions
|
Designation |
Tester |
Driver |
Strain |
Supplier |
Notes |
|
|
|
|
|
|
|
|
1-SSH |
Spinal cord |
Posterior 1/3 cerebral cortex |
Swiss Webster |
Pel Freeze |
|
|
2-SSH |
Spinal cord |
Posterior 1/3 cerebral
cortex |
Swiss Webster |
Pel Freeze |
Hybridizations in presence
of excess Plp1 |
|
3-SSH |
Spinal cord |
Visual Cortex |
C57Bl6J |
|
|
|
3-MOS |
Spinal cord |
Visual Cortex |
C57Bl6J |
|
|
|
4-SSH |
Spinal cord |
Visual Cortex |
C57Bl6J |
|
|
|
4-MOS |
Spinal cord |
Visual Cortex |
C57Bl6J |
|
|
Table I.
Classification scheme for subtractions 1-4 and subsequent mirror
orientation selections. Nomenclature is
as follows: 1-SSH represents subtraction 1 using suppression subtractive
hybridization, etc. 3-MOS represents subtraction 3-SSH subsequently analyzed by
mirror orientation selection.
Genes up-regulated in spinal cord versus visual cortex
|
Gene Abbreviation |
Subtraction Number |
|
|
Protein Category |
|||||
|
Probable Source |
1-SSH |
2-SSH* |
3-SSH |
3-MOS |
4-SSH |
4-MOS |
Total |
Percent |
|
|
Glial (16 unique) |
|
|
|
|
|
|
|
|
|
|
Ckb |
2 |
|
|
|
|
|
2 |
1.40 |
enzyme |
|
Cnp1 |
|
1 |
|
|
|
|
1 |
0.70 |
enzyme |
|
Ugt8 |
|
|
|
1 |
|
|
1 |
0.70 |
enzyme |
|
Cryab |
|
|
|
1 |
|
|
1 |
0.70 |
hs protein |
|
Atp1b2 |
|
|
|
2 |
|
|
2 |
1.40 |
membrane |
|
Dbi/EP/ACBP |
|
|
|
|
1 |
|
1 |
0.70 |
membrane |
|
Mag |
|
1 |
|
|
|
|
1 |
0.70 |
membrane |
|
Pmp22 |
|
|
|
|
1 |
|
1 |
0.70 |
membrane |
|
Tspan2 |
|
|
|
|
1 |
|
1 |
0.70 |
membrane |
|
Gfap |
|
|
1 |
|
|
|
1 |
0.70 |
structural |
|
Mbp |
2 |
|
|
|
|
|
2 |
1.40 |
structural |
|
Mobp |
|
|
|
3 |
|
|
3 |
2.10 |
structural |
|
Plp1 |
11 |
* |
3 |
1 |
9 |
8 |
32 |
22.38 |
structural/neuron
survival |
|
Fth1 |
|
|
8 |
3 |
|
1 |
12 |
8.39 |
transport
Fe |
|
Trf |
|
|
1 |
|
|
|
1 |
0.70 |
transport
Fe |
|
Kcnj10 |
|
|
|
|
1 |
|
1 |
0.70 |
transport
K |
|
|
|
|
|
|
|
|
|
44.06 |
|
|
Neural
(19 unique) |
|
|
|
|
|
|
|
|
|
|
Ndrg4 |
|
|
|
1 |
|
|
1 |
0.70 |
differentiation/axon
survival |
|
Plekhb1 |
|
|
|
|
1 |
|
1 |
0.70 |
signaling |
|
Dnci2 |
|
|
|
|
2 |
|
2 |
1.40 |
motor
protein |
|
Ywhae |
|
|
1 |
|
1 |
|
2 |
1.40 |
signaling |
|
SSB1/4930422J18Rik |
|
1 |
|
|
|
|
1 |
0.70 |
signaling |
|
Nefh |
|
|
3 |
2 |
2 |
3 |
10 |
6.99 |
structural |
|
Nef3 |
3 |
|
|
|
|
|
3 |
2.10 |
structural |
|
Slc12a2 |
|
|
1 |
|
1 |
|
2 |
1.40 |
transport
Na+K+Cl- |
|
2900054P12Rik |
|
1 |
|
|
|
|
1 |
0.70 |
unknown |
|
AI841971 |
|
|
1 |
|
|
|
1 |
0.70 |
unknown |
|
2610024G14Rik |
|
|
1 |
|
|
|
1 |
0.70 |
unknown |
|
clone:6330420E11 |
|
|
1 |
|
|
|
1 |
0.70 |
unknown/hypothetical
protein |
|
Phactr4 |
|
|
1 |
|
|
|
1 |
0.70 |
unknown/synaptosomes |
|
Aak1 |
|
1 |
|
|
|
|
1 |
0.70 |
vessel
endocytosis clatherin |
|
BC019977 |
|
|
|
|
1 |
|
1 |
0.70 |
vessel
exocitosis |
|
Gpr37l1 |
|
|
|
|
1 |
|
1 |
0.70 |
vessel/synaptic
& clathrin |
|
Nsf |
|
1 |
|
|
|
|
1 |
0.70 |
vesicle
fusion |
|
Scd1 |
|
|
|
|
|
1 |
1 |
0.70 |
enzyme
membrane |
|
spp1 |
|
|
|
1 |
1 |
1 |
3 |
2.10 |
matrix
extra cellular |
|
|
|
|
|
|
|
|
|
24.48 |
|
|
Undetermined
(22 unique) |
|
|
|
|
|
|
|
|
|
|
Cdc3716 |
|
|
|
|
1 |
|
1 |
0.70 |
cell
cycle |
|
Ccni |
|
|
1 |
|
|
|
1 |
0.70 |
cell
cycle |
|
Anln |
|
|
|
|
1 |
|
1 |
0.70 |
cell
cycle |
|
Dlk1 |
|
1 |
|
|
|
|
1 |
0.70 |
differentiation |
|
Fanc1 |
2 |
|
|
|
|
|
2 |
1.40 |
DNA
repair |
|
Cept1 |
|
|
|
|
2 |
|
2 |
1.40 |
enzyme/
membrane |
|
Idi1 |
|
|
|
|
1 |
|
1 |
0.70 |
enzyme/lipid
metabolism |
|
Dld |
|
|
|
1 |
|
|
1 |
0.70 |
enzyme/mitochondrial
function |
|
Intergenic |
|
1 |
|
|
|
|
1 |
0.70 |
intergenic pseudo sim to
G3PDH |
|
Atp5c1 |
|
|
|
2 |
|
|
2 |
1.40 |
mitochondrial
ATP synthetase component |
|
Slc25a3 |
|
1 |
|
|
|
|
1 |
0.70 |
mitochondrial
phosphate carrier |
|
Lars2 |
|
4 |
3 |
1 |
3 |
|
11 |
7.69 |
mitochondrial
tRNA |
|
Cltc |
|
|
|
|
|
1 |
1 |
0.70 |
structural
endosome/lysosome |
|
Med31 |
|
|
1 |
|
|
|
1 |
0.70 |
transcription |
|
2810437L13Rik |
|
|
|
|
1 |
|
1 |
0.70 |
unknown |
|
Itm2a |
|
|
|
|
2 |
|
2 |
1.40 |
unknown |
|
D8Ertd325e |
|
|
1 |
|
|
|
1 |
0.70 |
unknown |
|
Dock10 |
|
1 |
|
|
|
|
1 |
0.70 |
unknown/cell
morphology |
|
Sparc |
|
|
1 |
|
|
|
1 |
0.70 |
unknown/cell
morphology/ |
|
2500002L14Rik |
|
1 |
|
|
|
|
1 |
0.70 |
unknown/metal
ion binding |
|
Ikbke |
|
|
1 |
|
|
|
1 |
0.70 |
unknown/transcription
control |
|
A930004K21Rik |
|
|
1 |
|
|
|
1 |
0.70 |
unknown/translation
initiation |
|
Zfybe20 |
|
|
|
|
1 |
|
1 |
0.70 |
unknown/zinc
finger/endosomal traffic |
|
|
|
|
|
|
|
|
|
25.87 |
|
|
Mitochondria
(3 unique) |
|
|
|
|
|
|
|
|
|
|
CYTB |
|
|
|
|
|
1 |
1 |
0.70 |
cytochrome c reductase
complex |
|
LA9 |
|
|
|
|
2 |
1 |
3 |
2.10 |
mitochondrial
gene |
|
Trnl1 |
|
|
|
|
|
2 |
2 |
1.40 |
tRNA |
|
|
|
|
|
|
|
|
|
4.20 |
|
Table II Listing of
genes represented in various SSH and MOS analyses in order of probable source followed
by category of product. The total number
of unique genes represented was 60 and the total number of sequences analyzed
was 142. Genes are identified by
accepted abbreviations. *indicates that
this subtraction was performed in the presence of excess Plp1-cDNA as described
in Materials and Methods.
Comparison of
Repeating Sequences by SSH and MOS
|
Gene |
Accession |
Unigene |
|
|||
|
Abbreviation |
Number |
Designation |
3-SSH |
3-MOS |
4-SSH |
4-MOS |
|
Plp1 |
NM_011123 |
Mm.1268 |
3 |
1 |
9 |
8 |
|
Fth1 |
NM_010239 |
Mm.1776 |
8 |
3 |
|
1 |
|
Lars2 |
NM_153168 |
Mm.276076 |
3 |
1 |
3 |
|
|
Nefh |
NM_010904 |
Mm.298283 |
3 |
2 |
2 |
3 |
|
spp1 |
BC057858 |
Mm.288474 |
|
1 |
1 |
1 |
|
rRNA-MT |
DQ106412 |
|
|
|
2 |
1 |
|
Ywhae |
NM_009536 |
Mm.234700 |
1 |
|
1 |
|
|
Slc12a2 |
NM_009194 |
Mm.4168 |
1 |
|
1 |
|
|
Total |
|
|
19 |
8 |
19 |
14 |
Table III. Comparison of frequency of most commonly appearing
genes in subtractions three and four (3-SSH and 4-SSH) with MOS frequencies
resulting from these subtractions (3-MOS and 4-MOS).
Figure Legends
Fig. 1 Example of subtraction efficiency as estimated by polymer chain reaction (PCR) employing primers specific for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) sampled at cycles 18, 22, 32 and 37 for forward unsubtracted (FU); forward subtracted (FS); reverse unsubtracted (RU) and reverse subtracted (RS) conditions on subtraction 2-SSH. Forward refers to cDNA from spinal cord; reverse refers to cDNA from visual cortex; unsubtracted and subtracted refer to cDNA before and after suppression subtractive hybridization. Subtraction efficiencies in excess of 1000 fold were obvious from the comparison of the appropriate band intensities.
Fig. 2 Northern blots of total RNA from 90-110 day old C57Bl6J male mice hybridized with radiolabeled probes against A: Proteolipid protein; B: Myelin basic protein; C: Myelin-associated glycoprotein and D: Ferritin heavy chain. Clones in figure 2 were selected on the basis of being associated with myelin. All myelin associated probes show up-regulation in spinal cord when compared to visual cortex.
Fig. 3 Northern blots of total RNA from 90-110 day old C57Bl6J male mice hybridized with radiolabeled probes against A: G-protein coupled receptor 37 like 1; B: Clone 6330420E11 RIKEN; C: RIKEN cDNA 2500002L14 gene and D: Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein. Clones in figure 3 were randomly selected. All probes show up-regulation in spinal cord when compared to visual cortex.
Acknowledgement
This work is supported by a bequest from the estate of
We would like to thank the members of the board of directors of Solomon Park Research Institute for their tireless dedication to the Institute and the ongoing research effort.