Attachments

[Guest guest]

I want a green lymie ribbon!!!!

[ ] Attachments

Pepi,

You shouldn't really be opening attachments, there is always the possibility

that it's infected with a computer virus. In fact, I have been considering

making it impossible for to send attachments. What do you guys

think about that? Would that be a good thing or a bad thing? My concern is

computer viruses. (A virus did go through not to long ago as an

attachment.) Most things that people attach can be put into an email

directly. Let me know if you have any strong feelings about this either

way.

Back to your question Pepi - I'm not sure why it's not working. Most of the

time when you (actually I) double click on the attachment it will ask you if

you want to open it or save it to disk. Clicking on Open should just open

the document if it's associated with a program you have on your computer.

For instance, if it's a Word document, it will automatically open it in

Word, if you have Word on your computer. A basic Text document will open in

a frame that looks like your email program. I don't know why it's asking

you for a drive. Wish I could be more helpful. Does anyone else on the

list understand this?

Are you getting a lot of attachments through ?

Robynn

May is Lyme Disease Awareness Month.

Support a Lymie, wear a lime-green ribbon.

Re: [ ] Fw: really scared now(was this all

K, Robyn, why cant I open most attachments? when i try it, a box pops up

asking which drive to use? help me! ty Pepi

_____

<1/4322/8/_/484634/_/959017423/>

<http://adimg./img/4322/8/_/484634/_/959017423/pr_drill_468_12k.g

if>

_____

is dedicated to Marta McCoy, the foundation behind what is

today.

Easy Reference:

Send a blank email message to:

-Subscribeegroups - Subscribe to the list through email

-Unsubscribeegroups - Unsubscribe from the list

-Digestegroups - Switch your subscription to a digest format

-Normalegroups - Switch your subscription to normal

Please send messages not related to Lyme disease to

-Offtopicegroups

Archives can be accessed at lyme-aid

<lyme-aid>

Please visit the chat room at chat/lyme-aid

<chat/lyme-aid>

[Guest guest]

I want a green lymie ribbon!!!!

[ ] Attachments

Pepi,

You shouldn't really be opening attachments, there is always the possibility

that it's infected with a computer virus. In fact, I have been considering

making it impossible for to send attachments. What do you guys

think about that? Would that be a good thing or a bad thing? My concern is

computer viruses. (A virus did go through not to long ago as an

attachment.) Most things that people attach can be put into an email

directly. Let me know if you have any strong feelings about this either

way.

Back to your question Pepi - I'm not sure why it's not working. Most of the

time when you (actually I) double click on the attachment it will ask you if

you want to open it or save it to disk. Clicking on Open should just open

the document if it's associated with a program you have on your computer.

For instance, if it's a Word document, it will automatically open it in

Word, if you have Word on your computer. A basic Text document will open in

a frame that looks like your email program. I don't know why it's asking

you for a drive. Wish I could be more helpful. Does anyone else on the

list understand this?

Are you getting a lot of attachments through ?

Robynn

May is Lyme Disease Awareness Month.

Support a Lymie, wear a lime-green ribbon.

Re: [ ] Fw: really scared now(was this all

K, Robyn, why cant I open most attachments? when i try it, a box pops up

asking which drive to use? help me! ty Pepi

_____

<1/4322/8/_/484634/_/959017423/>

<http://adimg./img/4322/8/_/484634/_/959017423/pr_drill_468_12k.g

if>

_____

is dedicated to Marta McCoy, the foundation behind what is

today.

Easy Reference:

Send a blank email message to:

-Subscribeegroups - Subscribe to the list through email

-Unsubscribeegroups - Unsubscribe from the list

-Digestegroups - Switch your subscription to a digest format

-Normalegroups - Switch your subscription to normal

Please send messages not related to Lyme disease to

-Offtopicegroups

Archives can be accessed at lyme-aid

<lyme-aid>

Please visit the chat room at chat/lyme-aid

<chat/lyme-aid>

[Guest guest]

Robyn, I rarely open attachments, however I wants to open the FYI and

another that I got today. Ty very much for your help. btw, some will open in

word or something else, others just ask what drive, go figure! Pepi

[Guest guest]

In a message dated 5/22/00 1:44:34 PM Eastern Daylight Time,

nebneb@... writes:

<< In fact, I have been considering making it impossible for to

send attachments. What do you guys think about that? Would that be a good

thing or a bad thing? >>

I think thats a good thing !

[Guest guest]

From: " Pepi " <rod@...>

Robyn, I rarely open attachments, however I wants to open the FYI and

another that I got today. Ty very much for your help. btw, some will open

in

word or something else, others just ask what drive, go figure! Pepi

----------

Pepi,

It could be the computer is asking if you want to open what may be a " url

address " . I was trying to remember if the FYI came from a website? It is

coming to you in HTML format and possibly you cannot view HTML in your mail

program? I copied and pasted for you below.

_____________________________________

To All,

FYI.

Larry NV

The bdr Gene Families of the Lyme Disease and Relapsing Fever Spirochetes

Potential Influence on Biology, Pathogenesis, and Evolution

M. ,* A Carlyon,? Theisen,? T. Marconi*

* Medical College of Virginia at Virginia Commonwealth University, School

of

Medicine, Richmond, Virginia, USA

? Yale School of Medicine, Yale University, New Haven, Connecticut, USA

? Statens Serum Institute, Copenhagen, Denmark

[Emerging Infectious Diseases 6(2), 2000. Centers for Disease Control]

Abstract

Species of the genus Borrelia cause human and animal infections, including

Lyme disease, relapsing fever, and epizootic bovine abortion. The borrelial

genome is unique among bacterial genomes in that it is composed of a linear

chromosome and a series of linear and circular plasmids. The plasmids

exhibit significant genetic redundancy and carry 175 paralogous gene

families, most of of unknown function. Homologous alleles on different

plasmids could influence the organization and evolution of the Borrelia

genome by serving as foci for interplasmid homologous recombination. The

plasmid-carried Borrelia direct repeat (bdr) gene family encodes

polymorphic, acidic proteins with putative phosphorylation sites and

transmembrane domains. These proteins may play regulatory roles in

Borrelia.

We describe recent progress in the characterization of the Borrelia bdr

genes and discuss the possible influence of this gene family on the

biology,

pathogenesis, and evolution of the Borrelia genome.

Introduction

Species of the genus Borrelia cause human and animal infections[1]. In

North

America, Lyme disease and endemic relapsing fever pose the greatest threat

to human health and have received the most attention of the borrelial

diseases. Approximately 14,000 cases of Lyme disease are reported in the

United States each year; however, the actual number of cases may be 10-fold

higher[2]. Lyme disease was not recognized as a distinct clinical entity in

North America until the 1970s[3]. The causative agent, a previously

uncharacterized spirochete transmitted through the bite of infected ticks

of

the Ixodes ricinus complex (I. scapularis in the Northeast and Midwest and

I. pacificus on the West Coast)[4,5], was classified in the genus Borrelia

and named B. burgdorferi. With the emergence of Lyme disease and the

identification of its etiologic agent, Borrelia research focused on the

development of reliable Lyme disease diagnostic assays and vaccines, and

the

phenotypic and genotypic diversity of Borrelia was thoroughly analyzed.

Through modern molecular taxonomic techniques, several newly described

species of Borrelia have emerged as possible causative agents of Lyme

disease or at least as agents genetically related to B. burgdorferi[6-15].

The B. burgdorferi sensu lato complex is composed of the following species:

B. turdae, B. tanukii, B. bissettii, B. valaisiana, B. lusitaniae, B.

bissettii, B. andersonii, B. japonica, B. garinii, and B. afzelii. Of

these,

B. burgdorferi, B. garinii, and B. afzelii are the dominant species

associated with infection in humans.

Relapsing fever has been studied not only for its impact on human health

but

also as a model system for antigenic variation. There are two general forms

of relapsing fever, epidemic (louse borne--Pediculus humanus) and endemic

(tick borne--Ornithodoros spp.)[1]. Epidemic relapsing fever tends to be

associated with poor living conditions and social disruption (famine and

war) and is rare in the United States. Endemic relapsing fever is more

prevalent, predominantly in the western regions. Three closely related

Borrelia species, B. hermsii, B. turicatae, and B. parkeri, are associated

with this disease. Hallmark features of relapsing fever include cyclic

fever

and spirochetemia. The molecular basis for these features can be attributed

to the differential production of dominant variable surface antigens of the

Vmp protein families[16]. The 40 or so plasmid-carried vmp related genes in

the B. hermsii genome are expressed only one at a time. A single expression

locus exists, and genes not at this site lack a promoter element and are

therefore not transcribed[17]. The expressed Vmp becomes primary target of

a

vigorous humoral immune response that kills most of the spirochetal

population. However, at a frequency of approximately of 1 x 10-3 to 1 x

10-4

per generation, the identity of the expressed Vmp changes[18] through gene

conversion[19]. The net effect of this nonreciprocal event is to replace

the

gene located in the expression locus with one that was previously silent.

The production of a new antigenically distinct Vmp allows evasion of the

humoral immune response. This ongoing change in Vmp synthesis allows the

relapsing fever spirochete population to reestablish itself in the host,

thus leading to spirochetemia and the relapse of fever. Antigenic variation

systems have also been identified in the Lyme disease spirochetes; however,

they appear to exert a more subtle effect[20].

While clinical relapsing fever and Lyme disease differ from each other in

many ways, their causative agents share many similarities at both the

biologic and genetic levels. At the biologic level, they are host

associated

and undergo similar environmental transitions in the course of cycling

between mammals and arthropods. In view of the distinctly different

characteristics of these environments, the spirochetes must be able to

adapt

rapidly. Evidence suggests that the relapsing fever and Lyme disease

spirochetes use related proteins to adapt to or carry out similar functions

in changing environments. For example, homologs of the plasmid-carried ospC

gene of the Lyme disease spirochetes are carried by several other Borrelia

species, including the relapsing fever spirochetes[21]. Both ospC and its

relapsing fever spirochete homolog (vmp33) are selectively expressed during

the early stages of infection, which suggests that they play a common

functional role[22,23]. The B. burgdorferi Rep or Bdr protein family is

also

distributed genuswide. Members of this polymorphic protein family possess

highly conserved putative functional motifs and structural properties,

which

suggests that they may also carry out an important genuswide role[24,25].

The Borrelia Genome

At the molecular level, a unique feature of Borrelia is the unusual

organization and structure of their genome. Unlike most bacteria, which

carry their genetic material in the form of a single, circular DNA

molecule,

Borrelia have a segmented genome[26-28]. Most genetic elements carried by

these bacteria are linear with covalently closed termini or telomeres[27].

The telomeres are characterized by short hairpin loops of DNA[29]. If heat

denatured, these linear molecules relax to form a single-stranded circular

molecule. If reannealed, they base-pair upon themselves to form a

double-stranded linear molecule that by physical necessity possesses a

short

single-stranded hairpin loop at each telomere. Genetic elements of this

structure are rare in bacteria and are reminiscent of certain viral

genomes.

In B. burgdorferi (isolate B31), the largest of the linear genomic elements

is the 911-kb chromosome[30]. The chromosome carries 853 putative ORFs,

most

of which are thought to encode housekeeping functions. The remaining 12

linear and 8 circular genetic elements are plasmids. The plasmids might

best

be thought of as mini-chromosomes, since as a group they are indispensable

in situ and may carry genes encoding proteins involved in housekeeping

functions[31]. In addition, they may further deviate from the true

definition of a plasmid in that their replication may not be independent

and

may instead be tightly coordinated with the replication of the

chromosome[32,33].

Nearly 50% of the plasmid-carried ORFs lack homology with known sequences,

which suggests that their encoded proteins may define the unique biologic

and pathogenetic aspects of Borrelia[30]. Several of the proteins derived

from these plasmid-carried genes of unknown function are antigenic or

selectively expressed during infection, which indicates that they function

in the mammalian environment[20,34-37]. A striking feature of the

plasmid-carried ORFs is that they are organized into 175 paralogous gene

families of two or more members[30]. Hence, the DNA content of the plasmids

is highly redundant. Since the maintenance of DNA is energetically

expensive, it is likely that this redundant DNA is of biologic importance

to

Borrelia. The paralogous gene families of Borrelia have been the focus of

intensive research as they are thought to play important roles in

pathogenesis and to influence genome organization and

evolution[20,30,35,38-40].

Identification of Borrelia Direct Repear (bdr) Related Genes

The bdr gene family is a large, polymorphic, plasmid-carried, paralogous

gene family of unknown function that was originally identified in B.

burgdorferi[41,42]. Members of this gene family have been characterized in

several Borrelia species and isolates (Table 1) and have been assigned

various gene names[25,41-44] (Table 2).We have adopted the bdr designation

in the context of a nomenclature system[25], summarized below. Genes

belonging to the bdr gene family were first identified through the analysis

of repeated DNA sequences in B. burgdorferi sensu lato complex

isolates[41,42]. Seven nonidentical but closely related copies of a

plasmid-carried repeated element were identified in B. burgdorferi 297[42].

Three additional copies of this repeated sequence were further identified

in

B. burgdorferi 297[45]. These loci carry several ORFs that were designated

as rep+, rep-, LPA, LPB (the LP genes have recently been redesignated as

mlp

for multicopy lipoprotein[45]), rev, and the orfABCD operon (note: ORFs A

and B have been redesignated as blyA and blyB). Some of these genes,

particularly rep and mlp, exhibit allelic variation and encode polymorphic

proteins, the functions of which are under investigation. Focusing

specifically on the rep or bdr genes, the rep designation was originally

chosen to reflect a central repeat motif carrying domains in the deduced

amino acid sequences. The + and - designations were assigned to indicate

that the overlapping rep+ and rep- genes are located on opposing DNA

strands. Plasmid-carried repeated DNA sequences were also identified in B.

burgdorferi B31 and found to carry either all or a subset of seven ORFs,

designated A through G[41]. Of relevance to this discussion are the ORF-E

sequences that are rep or bdr homologs. A bdr-related gene was also

identified in B. afzelii DK1 and designated as p21[43]. B. afzelii causes

Lyme disease in Europe and Asia. The rep+, ORF-E, and p21 designations have

recently been replaced with bdr gene designations[24,25,44].

To assess and compare the composition and complexity of the bdr gene family

among species and isolates of the B. burgdorferi sensu lato complex,

restriction fragment length polymorphism (RFLP) patterns were determined

(Appendix). Genomic DNA digested with Xba1 was Southern blotted and probed

with an oligonucleotide targeting the bdr genes (Figure 1). A variable

number of hybridizing bands of different size were detected. These analyses

demonstrate that extensive bdr gene families are carried by B. burgdorferi

sensu lato complex isolates and that the RFLP patterns vary at the inter-

and intraspecies level. Hybridization analyses of other Borrelia species

showed that they also carry bdr-related gene families[24,25,46].

bdr-related

genes have been detected by hybridization in B. turicatae, B. hermsii, B.

parkeri, B. coriaceae, and B. anserina[25,46]. Isolates of these species

also exhibit substantial variation in their bdr RFLP patterns at the

intraspecies level. Table 1 lists the Borrelia species that carry

bdr-related genes and indicates the methods by which these genes or

proteins

were detected.

Figure 1. Restriction fragment length polymorphism pattern analysis of the

rep or bdr genes of the Lyme disease spirochetes. Total DNA, isolated from

Borrelia cultures, was digested with Xba1, fractionated by electrophoresis,

and transferred onto membranes for hybridization. Hybridization was

performed by the bdrAB-R1 oligonucleotide (46). The species and isolates

analyzed are indicated above each lane. MW markers in kb are indicated.

Figure 2. General organization of two bdr loci in Borrelia turicatae and B.

burgdorferi. The gene arrangement depicted for B. turicatae was determined

through cloning and sequence analysis of a 2,217 base-pair XbaI restriction

fragment. The arrangement for the bdr-carrying locus of B. burgdorferi was

previously determined through the sequencing of the B. burgdorferi B31

genome[30]. The arrows indicate the direction of transcription. Genes

exhibiting homology are indicated by similar shading or hatch marks. Genes

indicated by unfilled arrows are not homologous. The numbering is indicated

for scale and is not indicative of the positioning of these genes on the

plasmids that carry them.

Sequences flanking some bdr alleles also appear to be distributed genus

wide. Some bdr alleles of B. turicatae, B. parkeri, and B. hermsii are

flanked by genes that are homologs of genes carried by the Lyme disease

spirochetes[24,25]. As a specific example, the B. turicatae bdrA1 gene is

flanked by ORFs that are homologs of the BBG34 and BBG30 genes of B.

burgdorferi[24,25]. In the Lyme disease spirochetes, BBG34 is part of a

three-member paralogous gene family, while BBG30 is a single-copy gene[30].

Located between BBG30 and BBG34 is BBG33, a member of the bdr gene family

(recently redesignated as bdrF2)[25]. Although these divergent Borrelia

species carry related genes, their organization differs[24], which

indicates

that rearrangement has taken place in the ancestral plasmid that carried

these homologs.Figure 2 compares the organization of two bdr loci from B.

turicatae and B. burgdorferi.

Evolutionary Analyses of bdr-Related Sequences: Revised Nomenclature for

the

Bdr-Related Proteins

To simplify the complicated nomenclature of bdr-related genes, a bdr

nomenclature system has been developed that assigns gene names on the basis

of phylogenetic relationships inferred from comparative analysis of

genetically stable regions of the bdr genes[25]. This system, which is

applicable genuswide, allows for a ready assessment of relationships among

bdr paralogs and orthologs. The rationale for this system stemmed from the

results of a comprehensive evolutionary analysis of >50 bdr-related

sequences from five Borrelia species that demonstrated that bdr sequences

are organized into six distinct subfamilies, designated A through F[25].

Subfamilies are not necessarily species specific; some contain bdr alleles

from different Borrelia species[25]. Since members of a given subfamily are

closely related to one another with identity values for the N terminal

domain being >95%, each member is assigned the same gene name designation,

and paralogs are distinguished by a numerical subscript. In B. turicatae

OZ-1, two bdr subfamilies, bdrA and bdrB, contain at least four and five

members, respectively[24]. Members of the bdrA subfamily are designated

bdrA1, bdrA2, bdrA3, and bdrA4, while members of the bdrB family are

designated bdrB1 through bdrB5. This revised Bdr nomenclature scheme was

modeled after that proposed for bacterial polysaccharide synthesis

genes[47]

and is in accordance with the nomenclature guidelines established by

Demerec[48].

The subfamily affiliation of bdr genes can be readily determined through

comparative sequence analyses of the amino acid segment preceding the

polymorphic repeat motif region of these proteins (described in detail

below)[25]. Relationship assessments based on the genetically stable N

terminal domain (vs. complete sequences) are preferable because the

calculated evolutionary distances and clustering relationships are not

artificially skewed by the variable number of repeat motifs present in the

repeat motif domain. Since the genetically unstable repeat motif domain

comprises as much as 50% of the total coding sequence in some alleles, it

can have a substatial impact on inferred relationships. In addition,

extensive sequence variation in the carboxyl termini of the Bdr proteins at

the inter-species level makes it difficult to align this domain with

confidence, which further influences the inferred relationships.

bdr evolutionary analyses show that Borrelia species carry members of at

least two bdr subfamilies[25,44]. In fact, B. burgdorferi carries three

distinct subfamilies. Multiple Bdr subfamilies in diverse Borrelia species

suggest that there has been selective pressure to maintain multiple bdr

alleles and bdr genetic diversity. This genetic diversity may increase the

functional diversity of the Bdr proteins.

Molecular Features and Physical Properties of the Bdr Proteins

While early analyses of Borrelia bdr genes demonstrated their multicopy

nature[41,42,46], the full extent of the complexity of the bdr gene family

in the Lyme disease spirochetes was not fully recognized until the B.

burgdorferi genome sequence was determined[30]. B. burgdorferi B31 was

found

to carry 17 distinct bdr-related genes (and one truncated variant)

distributed among different linear and circular plasmids. B. turicatae,

which carries at least nine different bdr alleles, carries these genes

exclusively on linear plasmids[24,25,46]. Other relapsing fever spirochete

species (B. parkeri and B. hermsii) are similar to the Lyme disease

bacteria

in that they carry bdr genes on both linear and circular plasmids[25]. In

the Lyme disease spirochetes each of the 32-kb circular plasmids, with the

exception of plasmids M and P, carry two different bdr genes separated by

seven or eight ORFs. Each of these circular plasmids carries one bdrD

subfamily member and one bdrE subfamily member. The maintenance of genes

belonging to different subfamilies on a single plasmid is consistent with

the possibility that each carries out a different function. In contrast, in

the Lyme disease spirochetes, the bdrF subfamily members are localized to

linear plasmids with only a single bdr gene per plasmid. These observations

suggest that there has been selective pressure to maintain the association

of specific subfamilies with specific types of plasmids. Less is known

about

the bdr-carrying plasmids and the organization of the bdr genes and

subfamilies in the relapsing fever borreliae. However, as in the Lyme

disease spirochetes, in B. turicatae most bdr-carrying plasmids carry two

bdr genes, one from subfamily bdrA and one from subfamily bdrB[24].

Figure 3. (click image to zoom) Key features and putative functional

domains

of the Bdr proteins. The schematic depicts a prototype Bdr protein with the

characteristics of each domain indicated. The abbreviation, ID%, is for

percentage amino acid identity at either the inter- or intra-family level

as

indicated in the figure. Standard amino acid abbreviations are used in the

figure to denote the conserved C-terminal lysine (K) or asparagine (N)

residues, which are thought to be exposed in the periplasm and the

cytoplasmically located core tripeptide of the repeat motif

(lysine-isoleucine-aspartic acid; KID).

The sequence of more than 50 bdr alleles from five different Borrelia

species has been determined (Table 2)[24,25,41-43,46]. These extensive

comparative sequence analyses led to the identification of conserved

features that provide insight into the possible biologic roles of the Bdr

proteins. For example, all bdr alleles carry centrally located repeat motif

domains (Figure 3). Although conserved in sequence, these domains vary in

length among alleles as a result of varying numbers of the repeat motif.

The

core tripeptide of the repeat is the sequence KID. The repeat motifs encode

consensus casein kinase 2 phosphorylation (CK2P) motifs of the sequence

T/SKID/E[43]. While it may appear somewhat paradoxical for bacteria to

carry

casein kinases, casein kinase is a descriptive term broadly applied to at

least two classes of ubiquitous protein kinases for which the substrates

may

include various enzymes and noncatalytic proteins involved in important

cellular regulatory functions[49]. Most proteins phosphorylated by CK2-like

kinases are highly acidic, as are the Borrelia Bdr proteins (isoelectric

points between 5 and 6). The phosphorylation site in CK2P motifs is either

the Ser or Thr residue of the motif. Although histidine kinases have been

known to exist in some bacteria, it has been widely held that bacteria lack

Ser - Thr kinases. However, Ser -Thr kinases have recently been identified

in several bacterial species, including Myxococcus, Anabeana, Freymella,

Yersinia, and Streptomyces[50]. Most importantly, analysis of the B.

burgdorferi genome sequence identified a putative Ser - Thr kinase

designated BB0648[30,50]. This ORF carries a domain that exhibits homology

with the active site of Ser - Thr kinases. B. burgdorferi also carries a

homolog of the PPM family of eucaryotic protein Ser - Thr

phosphatases[30,50]. The presence of these genes in B. burgdorferi suggests

that the Borrelia possess the machinery necessary for Ser - Thr

phosphorylation and dephosphorylation.

Another important conserved feature identified through sequence analyses is

the hydrophobic carboxyl terminal domain of approximately 20 amino acids.

Computer analyses conducted with the TMpred program indicate that this

domain has a high propensity to form a transmembrane helix[24,25]. The

Tmpred values for the 20 aa C-terminal domains are 2,000 to 2,600. A value

of 500 or greater is considered significant[24,25]. Comparison of the Bdr

putative transmembrane domain sequences from the Lyme disease spirochetes

with those from the relapsing fever spirochetes indicates that, while there

is conservation in physical properties, there is essentially no

conservation

of primary sequence. However, sequence conservation does exist at the

subfamily level[24,25]. Since the Bdr proteins lack an obvious export

signal, membrane association would most likely be with the spirochetal

inner

membrane, with the rest of the protein, which is hydrophilic, extending

into

the cytoplasm. The terminal residue of the protein is in almost all cases a

positively charged amino acid (lysine or asparagine). This residue could

extend into the periplasm and serve to anchor the Bdr proteins to other

cellular components, such as the peptidoglycan.

Immunologic Analyses of the Bdr Proteins

The presence of multiple bdr alleles and bdr subfamilies within isogeneic

populations has prompted speculation that there may be differential

expression at either the subfamily or individual allele level, possibly in

response to environmental stimuli[46]. Limited studies of bdr expression

and

production, based on either mRNA detection or immunoblot analyses, have

been

performed. Porcella et al.[42] used Northern hybridization to determine if

expression of B. burgdorferi bdr-related genes occurs during cultivation in

the laboratory under standard culture conditions (33°C in BSK media). Bdr

transcripts were not detected by this approach. Similarly, in an earlier

analysis, we also conducted Northern hybridization experiments to assess

bdr

expression[46]. We detected expression of B. turicatae OZ1 bdrA subfamily

members in bacteria cultivated under standard laboratory growth

conditions[46]. However, when reverse transcriptase (RT)-PCR methods were

applied, transcription of a single bdrA allele was detected[46]. B.

turicatae OZ-1 was later demonstrated to carry at least nine bdr alleles,

four of which belong to the bdrA subfamily. Analysis of the sequence of

these alleles showed that all four should have been readily amplified by

the

RT-PCR primer set because of the conservation of the primer binding

sites[24]. The lack of detection of transcript derived from these alleles

suggested that only a subset of the bdr A subfamily alleles is expressed.

This raised the possibility that other bdr alleles are either nonfunctional

genes or their expression requires different environmental stimuli. The

transcriptional expression of the bdrB subfamily has not been specifically

assessed. Thorough transcriptional analyses using allele-specific probes

and

primers are an important step, since they allow specific assessment of the

expression of individual bdr alleles under differing environmental

conditions. In addition, analyses of the upstream DNA sequences of

individual bdr alleles and their genomic location may elucidate the

molecular basis for bdr transcriptional regulation.

Figure 4. Immunoblot analyses demonstrating the variation in Bdr protein

expression in Borrelia species and isolates. Bacteria were cultivated and

prepared for analysis as described in the methods. Proteins were

fractionated by SDS-PAGE, immunoblotted and screened with

anti-BdrF1-B.afzelii DK1 antisera. The species and isolates analyzed are

indicated above each lane in panels A, B and C. The migration positions of

the protein standards are indicated in each panel.

Immunologic analyses have provided a somewhat different overall picture

regarding Bdr production. Immunologic analyses described in this report and

elsewhere[44] demonstrate that several members of the bdr gene family are

expressed during in vitro cultivation. We conducted a comprehensive

analysis

of the expression of Bdr proteins among Borrelia species. When antisera

raised against recombinant B. afzelii BdrF1[24] were used in immunoblot

analyses, several immunoreactive proteins were detected in cell lysates of

all Borrelia species tested (Figure 4). The only exception was B. anserina,

a causative agent of avian spirochetosis. Although bdr-related sequences

have been detected in B. anserina by hybridization techniques[46],

immunoreactive proteins were not detected in immunoblot analyses.

Additional

analyses are required to determine if this indicates absence of

translational expression or the lack of epitope conservation in this

species. In any event, the fact that immunoreactive bands were not detected

in this species attests to the specificity of the anti-Bdr antisera. As a

further demonstration of the specificity of the antisera and to highlight

the fact that the Bdr proteins are unique to Borrelia, a cell lysate of

Leptospira interrogans was included in the immunoblot analyses.

Immunoreactivity with proteins in the Bdr size range was not observed with

the anti-Bdr antisera in this spirochete species. Borrelia species that

expressed immunoreactive proteins included B. garinii, B. burgdorferi, B.

turdae, B. tanukii, B. japonica, B. valaisiana, B. afzelii, B. coriaceae,

B.

bissettii, B. miyamotoi, B. parkeri, B. hermsii, and B. turicatae (Table

1).

Particularly striking was the extensive variation in the number and

molecular weight of the immunoreactive proteins expressed, with up to 12

distinct Bdr proteins detected. Variation in expression patterns was

observed at both the inter- and intraspecies level. Analysis of three B.

burgdorferi isolates (B31G, cN40, and CA12) demonstrated variability in

both

the size and number of expressed Bdr proteins. Isolate B31G has been

demonstrated by genomic sequencing to carry 18 distinct bdr alleles.

Immunoblot analyses show that not all alleles are expressed during in vitro

cultivation; therefore, some alleles may be differentially regulated.

The broad immunoreactivity of the antisera with diverse Borrelia species

indicates that some epitopes are conserved genuswide. In view of the

sequence divergence in the N and C terminal domains of the Bdr proteins

derived from different subfamilies, it is likely that the cross-reactive

epitopes reside in the conserved repeat motif region. Consistent with this,

computer analyses of the repeat domain of all determined Bdr protein

sequences predict them to be alpha helical and to have a surface exposed on

the protein and a positive on-Wolf antigenic index[24,25,44]. The

conservation and synthesis of these polymorphic proteins in such a diverse

group of Borrelia species suggest that they play an important role in

Borrelia biology genuswide.

The Bdr Proteins and Borrelia Biology: An Overview

Bdr genes and extensive bdr gene families have now been identified and

characterized in several diverse Borrelia species[24,25,42-44,46].

Comparative sequence analyses, which have identified conserved putative

functional domains, have provided the basis for the development of

hypotheses regarding Bdr function and cellular location. The Bdr proteins,

which lack known consensus export signals, are likely anchored to the

cytoplasmic membrane through their conserved, hydrophobic, putative

transmembrane spanning domain. The C-terminal positively charged amino acid

may be exposed to the periplasm, where it may interact with other cellular

components that may include the peptidoglycan. The repeat motif domain,

which is predicted by computer analyses to be hydrophilic and surface

exposed on the protein, likely extends into the cytoplasm. The conserved

repeat motif domain that carries the putative Ser - Thr phosphorylation

motifs may then be accessible for phosphorylation or to interact with other

cytoplasmic proteins or DNA to form a membrane anchored complex. As with

numerous other proteins, phosphorylation and dephosphorylation could play a

regulatory role, perhaps in signaling or sensing.

Multiple polymorphic bdr alleles may increase the functional range and

diversity of the Bdr proteins. Functional partitioning among Bdr proteins

could offer a possible explanation of why Borrelia expend such biologic

energy to maintain these genes in large gene families and express variants

of these proteins. The homology among bdr alleles may also allow or lead to

the continual modification of these genes through homologous recombination.

In fact, the variable nature of the repeat motif region, which is clearly

not evolutionarily stable, has likely arisen from slipped-strand

mispairing,

recombination, or rearrangement. In view of the extensive genetic

redundancy

of the plasmid component of the Borrelia genome, recombination in and among

related sequences on different plasmids could affect the organization and

evolution of the genome and ultimately host-pathogen interaction. Inter- or

intra-plasmid exchange of DNA sequences could provide a mechanistic basis

for the extensive genetic variability that has been widely described for

Borrelia plasmids[28,29,51-59]. In spite of the apparent necessity for at

least most of the plasmids for survival, as inferred from their ubiquitous

distribution among Borrelia isolates, these bacteria are able to tolerate

remarkable genomic variability. Diversity in the plasmids and the genes

they

carry may actually be exploited as a tool for phenotypic diversity and

rapid

environmental adaptation.

[Guest guest]

Ditto here.

Kathy

Re: [ ] Attachments

> In a message dated 5/22/00 1:44:34 PM Eastern Daylight Time,

> nebneb@... writes:

>

> << In fact, I have been considering making it impossible for to

> send attachments. What do you guys think about that? Would that be a

good

> thing or a bad thing? >>

> I think thats a good thing !

>

>

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[Guest guest]

my opinion is to allow attachements however all people should excersise

caution when opening any.....it is a personal thing....many people open all

they get i look at them .....especially if it is not a well know type....such

as doc...zi[....or jpg....i willl not open plus keep ur virus protection

updated....i have had no problems so far....( knock on wood)

Reid

[Guest guest]

not exactly sure what is going on with the mail, it looks as if the e mails

have pictures attached to them, and I was concerned for a while, but when I

check the box that says I know the sender of the e mail, there actually is no

picture attached to it, so its just fine.

Which sort of doesn't help, but it does?

In other words, with no attachment, can't be a virus, but this is odd, and I

do wish it would stop.

Ellen

[Guest guest]

Ellen,

I thnk the problem is that the original letter in the thread had a

picture attached and several people have just attached their reply to the

first one with the attachment. I have been getting the same message and

don't know how to detach it, but if everyone starts with a clean letter form

instead of just adding to the previous letter, it should disappear ( I hope).

Jackie

[Guest guest]

Dana - Your letter came through with the attachment on it. Try starting with

an empty letter form next time and see if that does the trick.

Jackie

[Guest guest]

Dana--

do you have an anti-virus program? I think you are the bug!!!!!!!!!!

Ellen

[Guest guest]

OK Jule I admit I hit the reply button . . .

You make a great argument (Jackie too) about starting a new e-mail to

respond to a post, BUT when people follow this advice I don't know what they

are talking about! I need at least a bit of the post that's being responded

to, to have a clue.

Yes I could scroll through messages and match up replies and original posts

.. . . but in point of practical fact, I don't very often as this is a chore.

(At least it was for about a week, last time you suggested posters not

include the post they are responding to, then everybody seemed to regress!

:-)

Whine, whine.

Kathy R. in Indiana

----- Original Message -----

From: <j.monnens@...>

> I will add my 2 cents about attachments. Many people simply hit reply

when they

> respond to an email from the list. For some of us, this means that we not

only

> receive the entire previous post, but also an attachment containing the

previous

> post and assorted other stuff. That is why I have asked people to please

> respond with a NEW message rather than simply hitting reply. We are less

likely

> to get viruses that way, and it will take up less space on the server as

well.

[Guest guest]

Kathy R:

What I suggest is cutting and pasting the portion of the post you are responding

to into your new message so everyone knows what you are responding to. That's

an ungainly sentence, but you get the idea.

Jule

[Guest guest]

HI Kathy:

I agree with you the train of posts really helps me. Aloha, Kathy

At 02:21 PM 10/02/2000 -0500, you wrote:

>OK Jule I admit I hit the reply button . . .

>

>You make a great argument (Jackie too) about starting a new e-mail to

>respond to a post, BUT when people follow this advice I don't know what they

>are talking about! I need at least a bit of the post that's being responded

>to, to have a clue.

>

>Yes I could scroll through messages and match up replies and original posts

>. . . but in point of practical fact, I don't very often as this is a chore.

>(At least it was for about a week, last time you suggested posters not

>include the post they are responding to, then everybody seemed to regress!

>:-)

>

>Whine, whine.

>

>Kathy R. in Indiana

[Guest guest]

A stupid, technical question for you Jule, is there any difference betw cut

and paste on a new e-mail, vs " reply " and then delete extraneous stuff? I'm

still a newbie, nearly flat learning curve where computers are concerned!

Kathy R. in Indiana

----- Original Message -----

From: <j.monnens@...>

> What I suggest is cutting and pasting the portion of the post you are

responding

> to into your new message so everyone knows what you are responding to.

That's

> an ungainly sentence, but you get the idea.

[Guest guest]

Kathy:

Replying and then deleting would probably work just as well. Might even be

easier for newbies.

Jule

PS I think the attachments and pictures are being sent by those who use

stationary rather than the regular email. But maybe not. As we used to say in

the ER -- GOK (God only knows.)

[Guest guest]

I have the MaCaffrey (sp?) that came with the computer but I don't have any of

the new updates. How would I know if I had a virus, since it doesn't seem to be

affecting me?

Dana in NC

elan214@... wrote:

> Dana--

>

> do you have an anti-virus program? I think you are the bug!!!!!!!!!!

>

> Ellen

>

>

> You may subscribe to the OCD-L by emailing listserv@... . In the

body of your message write: subscribe OCD-L your name. You may subscribe to

the Parents of Adults with OCD List at

parentsofadultswithOCD . You may access the

files, links, and archives for our list at

. Our list advisors are Tamar

Chansky, Ph.D., and Aureen Pinto Wagner, Ph.D. Our list moderators are

Birkhan, Kathy Hammes, Jule Monnens, Gail Pesses, Roman, and Jackie Stout.

Subscription issues, problems, or suggestions may be addressed to Louis Harkins,

list owner, at harkins@... .

[Guest guest]

HI Kathys,

Me too! I often refer to the message I'm replying to as I type, then

(with any luck) remember to delete the old message before I send.

Cheers,

Lesli

Kathy Hammes wrote:

>

> HI Kathy:

>

> I agree with you the train of posts really helps me. Aloha, Kathy

>

>

[Guest guest]

I second the motion, Jule. Cut and paste could solve the problem.

Jackie

[Guest guest]

Gayle,

I agree. I dont like attachments and usually dont bother reading them.

Shireen

>From: galye@...

>Reply- egroups

> egroups

>Subject: Re: [ ] To Gayle or Joan

>Date: Fri, 13 Oct 2000 18:34:41 EDT

>MIME-Version: 1.0

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>MHotMailBBB0D90D004140042A13D032905F75FD144; Fri Oct 13 15:36:44 2000

>Received: from [10.1.10.36] by ef. with NNFMP; 13 Oct 2000

>22:34:55 -0000

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>2000 22:34:50 -0000

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>with SMTP; 13 Oct 2000 22:34:49 -0000

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>a.ce.bcebffa (3952) for < egroups>; Fri, 13 Oct 2000

>18:34:41 -0400 (EDT)

>From sentto-165537-23765-971476491-shireen42 Fri Oct 13 15:40:21 2000

>X-eGroups-Return:

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>X-Sender: GALYE@...

>X-Apparently- egroups

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>

>Pat,

>I know I have answered when Cyndi asked questions but she posts so little.

>I

>was hoping she would post more often. You as well I have seen posts back to

>you. You have been putting your posts as attachments and I have been

>deleting

>those posts. I am afraid to open these because of past viruses and in

>support

>groups anyone can jump in use a name that we think will be safe. I received

>so I thought a birthday card last year. 4 days later my computer crashed.

>Took a week to fix it. I was lucky because I have a master computer wizard

>as

>a friend that fixed my computer for free, otherwise forget it I would have

>no

>computer. Since then I do everything in my power to avoid this. Tell Cyndi

>to

>post more.

>

> gayle/trans.6-99

>galye@... ^0^

>

> `

>

>

>

>

>

>

>

>

>

>

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[Guest guest]

i just was wondering if anyone else has to open attachments on certain

people's posts. originally it only seemed to happen with Robyn's mail because

of the text, but i notice now it happens with all of rose's post's, and

goodgirl (roberta) as well as a few others. anyone have an idea of how to

correct this on aol?

[Guest guest]

I recieve picture attachments too. And at first is was Robynn, now it is a lot of people. I upgraded to AOL 6.0 and now even my own messages come as picture attachments!!! I thought this would solve the problem but it hasn't. Happy (Maine)

[Guest guest]

picture attachments with no pictures.......yes, now your post has one too! pj

[Guest guest]

Rose....yours came as an attachment, but I think it is because of the banner at the bottom of the email......not your doing. Looks like it is a combination of things going on here!

Happy (Maine)....Off to the LDA conference in NJ!!!!!!

[Guest guest]

Hi Guys,

I know that my "Custom Signature" (currently "TOIL") at the bottom of each post is interpreted by some email systems as an "attachment." Don't know what to do to correct it, other than to ignore it. Also, the fact that I am using color for some of the text might also have changed things, if it wasn't this way before on your end.Just to test, I'm not going to use the signature on this post (it's optional). Let me know if that seems to be the cause of the problem in my case.Love ya, Rose

PJSNYDERNY@... wrote:

i just was wondering if anyone else has to open attachments on certain people's posts.

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