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The Glickman group's work with cultured cells was only recently initiated, but
substantial progress has been made in the construction of strains andd vectors
permitting mutations arising in mammalian cells to be transferred into bacteria
for further analysis, including DNA sequencing.

The Kunkel group has concentrated on the analysis of mutagenesis occurring
during the in vitro synthesis of DNA but detected by phenotypic alterations in
vivo and charcterized by DNA sequencing. The three major mammalian species of
DNA polymerase, characterized for their mutagenic propensities in a forward-
mutation system, show three distinct rates and specificities of mutation. A
major component of these differences is seen in a strong correlation between
the fidelity and the processivity of the polymerase; this discovery marks the
first insight into the nature of intrinsic polymerase accuracy, as distinct
from accuracy associated with the action of the 3'-exonucleases that occur in
prokaryotic polymerases but are absent from these mammalian polymerases. In a
second series of studies, specific kinds of DNA damage are being examined for
their mutagenic consequences. Abasic sites, such as those arising from spon-
taneous and/or Induced depuri nation, have been shown in the forward-mutation
assay to generate base-pair substitutions, double mutants, gene-conversion-
like mutational events and, at a lower rate, frameshift mutations. The most
common mutations are single base-pair substitutions. Most arise opposite
purines (especially guanine), and most involve the insertion of an adenine
residue opposite an apurinic site, thus generating transversions.



151



The Ripley group has concentrated on the analysis of frameshift mutagenesis
occurring in vivo in phage T4, followed by DNA sequence analyses of the muta-
tions. The wild-type and several mutant versions of the T4 DNA polymerase
cause distinctly different rates of frameshift mutagenesis in different
sequences and involving different changes. It has thus become possible to
delineate a number of distinct mechanisms of frameshift mutagenesis, many more
than known just a year or two ago. The classical type involves the addition
or deletion of a single residue from a simple repeating sequence, or the addi-
tion or deletion of a single block of resides from a repeating block. In
addition, however, single-base duplications are common outside of repeating
sequences. At least three types of complex mutations have been detected;
these involve multiple frameshift and base-pair substitution changes arising
in a single event. Some are due to the recently discovered quasi palindrome
correction mechanism. Others appear to be caused by a kind of gene-conversion
event acting over a considerable distance. A third group consists of frame-
shift mutations separated, by two or three bases, from a transversion; but
the mechanism that generates this class is still poorly understood. While the
above classes of frameshift mutagenesis operate spontaneously, it has recently
been found that proflavin-induced frameshift mutations tend to fall within
particular nucleotide octamers that share a strongly conserved sequence.
Again, the mutagenic propensity of these sequences is not yet understood.

The Schaaper group, originally a part of the Glickman group, is in the process
of establishing its independence. In addition to extensive participation in
the projects outlined as components of the Glickman program, Schaaper has been
working on EPR-like mutagenesis in vitro. This is a problem that has eluded
resolution in many excellent laboratories around the world over the past
decade. To date, procedures have been developed to detect mutations induced
by cell extracts acting upon a target gene in vitro, followed by in vivo
expression and, finally, characterization by DNA sequencing.

The Volkert group has concentrated on two aspects of DNA repair and mutagene-
sis in E. col i : the recF gene, and genes involved in the response to alkyla-
tion damage"^ Mutations in the recF gene affect both mutagenesis and
recombination, and a detailed analysis of the properties of recF mutations and
their extracistronic suppressors supports a model that assigns to the recF
protein a role in modulating two functions of the recA protein: its conver-
sion following UV irradiation of the cell to a protease, and its control over
the recF mode of recombination. (The srfA suppresor of a recF mutation
separates these two functions.) Genes involved in the response to alkylation
damage are being detected by means of insertion mutations, wherein the inser-
tion carries a portion of a lac gene whose activity can be monitored; mutants
are then selected in which the lac gene is activated specifically by alkyla-
tion damage, indicating that the recipient gene is induced by such damage. To
date, at least five such genes have been discovered, two being known pre-
viously in other contexts. The patterns of response of these genes are
complex, and are now being sorted out.

A number of personnel changes are occurring in FY-84. The Glickman and Ripley
groups are leaving the NIEHS. A major portion of the Glickman group, namely
the Schaaper group, is advancing to independent status within the Mutagenesis
Section. Finally, the Sugino group, which left the NIEHS two years ago, is
returning.



152



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER

ZOl ES 60098-05 LG



PERIOD COVERED

October 1, 1983 to September 30, 1984



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)

Mammalian Lactate Dehydrogenase Isozyme Analyses



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)

PI: Steven S.-L. Li Research Geneticist LG, NIEHS



COOPERATING UNITS (if any)



LAB/BRANCH

Laboratory of Genetics



SECTION

Eukaryotic Gene Structure Section



INSTITUTE AND LOCATION

NIEHS, NIH, Research Triangle Park, North Carolina 27709



TOTAL MAN-YEARS:



PROFESSIONAL:



OTHER:



CHECK APPROPRIATE BOX(ES)

D (a) Human subjects SI (b) Human tissues D (c) Neither

D (a1) Minors
□ (a2) Interviews



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)



Study continued under Project Number ZOl ES 61032-01 LG entitled "Structure-
function of mammalian lactate dehydrogenase isozymes".



PHS 6040 (Rev. 1/84) -.r-j GPO 904-917



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER

ZOl ES 60099-05 LG



PERIOD COVERED

October 1, 1983 to September 30, 1984



TITLE OF PROJECT (80 characters or less. Title must fit on orte line tietween the borders.)

Organization-regulation of mammalian lactate dehydrogenase genes



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)

PI: Steven S.-L. Li Research Geneticist LG, NIEHS



Others: Hiroshi Tsujibo
Kiyohito Yagi
Kayoko Fukasawa



Visiting Fellow
Visiting Fellow
Visiting Fellow



LG, NIEHS
LG, NIEHS
LG, NIEHS



COOPERATING UNITS (// any)



LAB/BRANCH

Laboratory of Genetics



SECTION

Eukaryotic Gene Structure Section



INSTITUTE AND LOCATION

NIEHS, NIH, Research Triangle Park, North Carolina 27709



TOTAL MAN-YEARS:

3.5



PROFESSIONAL:

2.0



OTHER:

1.5



CHECK APPROPRIATE BOX(ES)

n (a) Human subjects
D (al) Minors
n (a2) Interviews



21 (b) Human tissues D (c) Neither



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)

LDH-A cDNA clones from mouse and human have been isolated and sequenced. Mouse
pMLA 73 was found to contain the 393bp of coding region and 496 bp 3'
untranslated sequence including poly (A) tail. Four human cDNA clones consist
of sequences of approximately l,700bp, that is, the complete coding sequence
(999bp), the 5' (97bp) and 3' (565bp) untranslated regions. Several LDH genomic
clones have also been isolated and partially characterized from mouse and human
DNA libraries. Mouse genomic clone M15 appears to contain a functional gene of
at least 8Kb consisting of several exons and introns which have been sequenced.
The nucleotide sequence of a human LDH-A pseudogene has been determined. 12.9%
nucleotide differences were found between the pseudogene and LDH-A cDNA, and
their significant implications on mammalian mutations and molecular evolution
are observed. It is of interest to elucidate the genetic mechanism(s)
underlying the tissue-specific expression of three LDH genes in human and other
vertebrates.



PHS 6040 (Rev. 1/84)



154



GPO 904-917



ZOl ES 60099-05 LG



PROJECT DESCRIPTION

PROBLEM : Structural organization, regulation of expression and molecular evolu-
tion ot mammalian multigene families coding for ssDNA-binding lactate dehydroge-
nase proteins are being studied to understand the role of the several
polypeptide domains in the function and evolution of these proteins.

OBJECTIVES : In mammals lactate dehydrogenase isozymes A4 (muscle), B4 (heart)
and C4 (testes) are encoded by three different gene loci and the expression of
these three LDH genes is developmental ly regulated and tissue-specific.
Recently, the LDH isozymes were shown to be ssDNA-binding proteins. The
detailed knowledge about the genomic organization of this multigene family will
shed light on molecular aspects of their regulation of expression and molecular
evolution.

EXPERIMENTAL APPROACH AND SCIENTIFIC JUSTIFICATION : The genomic clones of three
LDH genes from human and mouse have been/will be isolated and their organization
and regulation of expression will be illustrated. Analysis of the DNA sequences
encoding the proteins and those flanking the structural genes will allow
accurate evaluations of genetic mutation events which perturb the regulatory
mechanisms as well as those which modify the protein coding sequence itself. In
humans the deficiencies of LDH-A or LDH-B subunits have been correlated with
different clinical features and the molecular nature of their genetic mutations
will be determined.

The di nucleotide CpG deficit appears to be a distinctive feature of mammalian
DNA and the methylated CpG sequences appear to control the tissue-specific gene
expression. The extent of DNA methyl ati on at the regulatory region of the human
LDH genes will be examined by the genomic blotting of placenta and sperm DNAs.
The methylated CpG sequences have also been suggested to be mutational hotspots
in mammalian DNA and the high frequency of polymorphism at Msp I and Tag I
restriction sites containing the CpG sequence have been observed in human DNA.

The expression of the LDH genes may be regulated by the stress factors such as
heat-shock, anaerobic-shock (anoxia, hypoxia), oxidative inhibitors, oxidizing
chemicals, etc. The cellular level of nucleotide pool such as ATP/ADP,
NAD+/NADH, AppppA, etc. may play an important role as signaling 'alarmones'.
Recently, LDH-K isozyme is found in many carcinomas and in cultured cells trans-
formed by the Kirsten murine sarcoma virus. The expression of this LDH-K seems
to be induced by anaerobic shock. The molecular basis for the expression of the
LDH-K and its possible relationship to the cancer will be studied.

RECENT ACCOMPLISHMENTS AND SIGNIFICANCE : Several mouse LDH-A cDNA clones have
been isolated and characterized. Mouse pMLA73 clone was found to contain the
393bp of protein-coding sequence and 496bp of the 3' untranslated region
including a poly (A) tail. The amino acid sequence deduced from its open
reading frame is in agreement with the COOH-terminal sequence of residues 201 to
331 of mouse LDH-A subunit determined independently by protein sequencing. This
cDNA clone was used as a probe to isolate nine genomic clones from a mouse gene
library. One of these genomic clones was shown to possess an insert of 13Kb.



155



ZOl ES 60099-05 LG

The authenticity of mouse LDH-A functional gene and the polarity of its
transcription were demonstrated by nucleotide sequencing. The exon-intron junc-
tion sites have thus far been found at amino acid no. 45, 83, 197, 236 and 278.
The complete nucleotide sequence of the last exon for COOH-terminal and 3'
untranslated region including the polyadenylation signal have been determined.
The alternating sequence of purine and pyrimidine in the Z-ONA and two Alu-
sequences have been found in the intron regions of this functional LDH-A gene
and their possible roles in the regulation of gene expression will be further
studied.

Eight LDH-A cDNA clones have been isolated and characterized from a human
fibroblast cDNA library. Four cDNA clones appear to contain nearly full-length
inserts and the complete nucleotide sequence of 1710bp consists of the protein-
coding sequence (999bp), the 5' (97bp) and 3' (565bp) untranslated regions and
poly(A) tail (49bp). These human LDH-A cDNA clones which were isolated from the
SV40-pBR322 based recombinants will permit the functional expression of nearly
full-length cONA sequences in mammalian cells. Thus, they will be extremely
useful for studying the relationship of the structure to diverse activities,
including ssDNA binding of LDH-A protein, through the in vitro directed muta-
genesis. Seventeen LDH-A like clones have also been isolated and characterized
from human genomic library. The nucleotide sequence of a processed, intronless
LDH-A pseudogene was determined. A comparison between human LDH-A cDNA and the
processed pseudogene sequences reveals 114 transitions, 65 transversions and 36
deletions/insertions. Only four out of the 25 CpG di nucleotides present in the
cDNA sequence remain unchanged, although the sequences possess 87.1% homology.
Further, one of the human genomic clones has also been shown to possess introns
and it may be a functional LDH-A gene.

Further restrictions mapping and partial nucleotide sequencing of nine mouse
genomic clones and seventeen human genomic clones as well as Southern blot ana-
lysis of mouse liver DNA and human placenta DMA appear to suggest the existence
of many LDH-like pesudogenes, and this may have significant implications in the
mechanisms of their retroviral origin and molecular evolution.

FUTURE RESEARCH PLANS : 1. The genomic clones of LDH-A, B and C functional
genes from human and mouse will be isolated and their structure determined by
analyses of nucleotide sequence, R-loop and heteroduplex. The potential control
(i.e. enhancer) and protein-coding sequences of different LDH genes will be com-
pared and analyzed in order to obtain the information on genetic regulation and
molecular evolution.

2. The molecular basis of human LDH-A and LDH-B deficiencies will be investi-
gated by genomic blot analysis using LDH gene probes and, possibly, by sequence
analysis of cloned mutant genes. The molecular nature of spontaneous and
induced missense mutations at mouse LDH-A gene locus will also be determined.

3. The developmental and tissue-specific expression of mouse LDH-A, B and C
genes will be studied by quantitative analysis of their mRNAs using gene-
specific probes. The in vitro cell-free transcription system and the in vivo
gene expression in cultured cells for human and mouse LDH genes will be



156



ZOl ES 60099-05 LG

developed. The in vitro mutagenesis of LDH genes will be used to study the
control of gene expression as well as structure-function of LDH proteins.

4. The extent of CpG methyl ation at the LDH-A gene will be compared among the
ONA samples isolated from different tissues of human and mouse. The nuclease
sensitivity of chromatin, especially, the 5' upstream regulatory region of the
LDH-A gene, will be studied. In view of its ssDNA-binding ability, the possi-
bility of autoregul ation at the transcription and translation levels of LDH
genes will be investigated.

5. The precise locations of LDH gene loci, especially LDH-C gene, in human and
mouse chromosomes will be determined by in situ hybridization and/or genomic
blot analysis of hybrid somatic cells. TRe possible relationship between the
expression of LDH genes and oncogenes (H-ras, K-ras & M-ras) will be
investigated.

PUBLICATIONS

Shimuzu, M. , Tsujibo, H. , Sharief, F.S., Tiano, H.F., Pan, Y.-C.E. , Jungmann, R.A.
and Li, S.S.-L.: Sequence analyses of cDNA and protein for maimialian lactate
dehydrogenase isozymes. Fed. Proc. 42: 1970, 1983.

Tsujibo, H. ,Yagi , K. , Tiano, H.F. , and Li, S.S.-L.: Cloning and characterization
of lactate dehydrogenase genes from mouse and human. Fed. Proc. 43: 1723, 1984.



157



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER

01 ES 60111-05 LG



PERIOD COVERED

October 1, 1983 to September 30, 1984



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)

Studies on the Role of Gene 43 DNA Polymerase in Frameshift Mutagenesis



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)

PI: L. S. Ripley Senior Staff Fellow LGM, NIEHS



Others: J. G. de Boer
A. B. Clark
K. M. Price
D. M. Ferber



Visiting Fellow

Biologist

Q

Q



LGM, NIEHS

LGM, NIEHS

LGM, NIEHS

LGM, NIEHS



(I



COOPERATING UNITS (if any)

Institute of Molec.
Univ. of Oregon
Eugene, OR



Biology



I AMP
NIH



LAB/BRANCH

Laboratory of Genetics



SECTION

Mutagenesis Section



INSTITUTE AND LOCATION



NIEHS, NIH, Research Triangle Park, North Carolina 27709



TOTAL MAN-YEARS;

3.8



PROFESSIONAL:

2



OTHER:

1.8



CHECK APPROPRIATE BOX(ES)

n (a) Human subjects
n (a1) Minors
n (a2) Interviews



n (b) Human tissues



IS (0) Neither



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)

Our ongoing investigation of frameshift mutagenesis in the T4 rll system reveals th€
specific enhancement of particular pathways of spontaneous frameshift mutagenesis by
different T4 DNA polymerases. Our studies now permit us to classify certain frame-
shift mechanisms by the genetic outcome (the nature of the DNA sequence change)
and thus to examine the role of DNA polymerase defects exhibited by mutator and
antimutator 14 DNA polymerases. For example, the mutator polymerase tsL98 produces
a large increase in frameshift mutations occurring in runs of A:T base pairs (100 t(|
200-fold) while producing no increase in the addition of single base pairs not
occurring in mononucleotide runs. Taken as a whole, our data strongly support the
notion that frameshifts of these two types occur via different mechanisms.
Sequences of spontaneous frameshifts offer support for two novel mechanisms of frame
shift mutation (Ripley, 1982; Ripley & Glickman, 1983; de Boer & Ripley, 1984).
The first mechanism depends upon the correction of imperfect palindromic DNA
sequences to more perfect palindromic DNA sequences. The second mechanism
generates frameshifts by converting an imperfectly homologous sequence to a
sequence that is perfectly homologous, but lies 256 base pairs down stream.
This intragenic conversion event occurs at a substantial frequency in certain
DNA polymerase mutant backgrounds examined. Sequences of proflavin-induced frame-
shift mutation in a wild type polymerase background demonstrate strong
sequence specificity. Our initial results identify 14/16 mutants lie within a
six base pair sequence. Not all of the mutations are identical (4 distinct
genotypes have been observed). This demonstrates a direct sequence effect on
occurence of frameshift mutations but may suggest the possibility of additional
factors that define the specificity of these frameshift sites. Further studies
in different mutant backgrounds and with other intercalating agents should per-
mit a distinction between elements of site specificity dependent on the site of
preferred mutagen binding from site specificity dependent upon site-specific DNA
metabolic events.



PHS 6040 (Rev. 1/84)



158



GPO 904-917



ZOl ES-60111-05 LG

METHODS EMPLOYED : Standard genetic manipulations of bacteriophage T4, using
mutants in the rll genes to measure mutation rates and frequencies (Ripley and
Shoemaker, 1983). Mutations in the DNA polymerase gene 43 were utilized to
alter replication fidelity (Ripley, 1982 for review). Sequencing of T4 rll fra-
meshifts has been accomplished by the shot gun cloning of T4 TaqI fragments in
M13 followed by dideoxysequencing. In vitro exploration of frameshift fidelity
has used specially constructed T4-M13 hybrid clones that permit the specific
identification of frameshift mutations in T4 rll sequences in M13 DNA.

MAJOR FINDINGS AND PROPOSED COURSE : A substantial fraction of spontaneous rll
frameshift mutants previously characterized genetically (Ripley and Shoemaker,
1983; Ripley, et al . 1983) has now been sequenced. Clear patterns of frameshift
mutations promoted by different DNA polymerases have emerged suggesting that
different aspects of DNA metabolism are altered in the various DNA polymerase
backgrounds. This conclusion is based on the finding that particular "classes"
of frameshift mutations are inhanced in particular DNA polymerase backgrounds.
The patterns of enhancement suggest that although one class may be enhanced by a
particular polymerase, similar enhancement of all classes does not occur. A
simple conclusion from this observation is that frameshift mutation frequencies
do not occur at enhanced frequencies in mutant DNA polymerase backgrounds as a
general consequence of poor DNA replication, but instead occur in a specific
manner implicating the active participation of the mutant DNA polymerase in the
initial production and/or the final fixation of the frameshift mutation.

Classes of frameshifts:

(1). The addition or deletion of a single base pair in a run of identical base
pairs, (e.g. 4 consecutive A:T pairs are mutated to either 5 or 3 consecutive
A:T pairs.)

A. These mutations are extremely frequent in tsL98 and tsL56 polymerase
backgrounds, but are infrequent in the tsL141 background when compared to the
wild type DNA polymerase. This result might suggest that proofreading of the
mismatched mutational heterozygote might be a major factor in the frequency of
this class of frameshift mutation since the tsL98 and tsL56 polymerases
demonstrate relatively low 3'-5' exonuclease activity in vitro, while tsL141
exhibits relatively high exonuclease activity compared to the wild type.

B. Mutations of this class are also enchanced in a tsL88 DNA polymerase
background. Since this mutator polymerase does not exhibit altered
proofreading, the enhancement of this class of frameshift mutations in this
background probably occurs by an alternative mechanism.

(2). The addition or deletion of multiple base pairs as a consequence of two
nearby homologous or nearly homologous DNA sequences.

A. The most frequently recovered mutation of this type in our DNA target
results in the addition or deletion of 8 bp within the sequence 5'-ATTGGCTGATTGGC-3'



159



ZOl ES-60111-05 LG

leaving either 5'-ATTGGCTGATTGGCTGAnGGC-3' or 5'-ATTGGC-3' . Such a mutation is
likely to arise through a misalignment of one copy of the ATTGGC sequence on the
complement of the other copy, the direction of misalignment relative to DNA
synthesis determines whether the outcome is + or -8 bp. We detect no large dif-
ference in the frequency of additions versus deletions in any DNA polymerase
background.

B. The frequency of enhancement of this class of frameshift mutation is
significant in the mutator DNA polymerase backgrounds, but is substantially
smaller than the addition or deletion of single bases in runs of that base
described above. For example, tsL98 increases the frequency of the single base
addition or deletions by 100 to 200-fold, while increasing the frequency of + or
-8 bp in the target described here only 3 to 7-fold.

C. Other deletions or duplications have been found to correlate with the
presence of repeated sequences at their end points. In some cases these repeats
are not perfectly homologous. In one deletion the repeat has 12 of 17 homolo-
gous base pairs.

(3). The duplication of single base having nearest neighbors that are different
from that base. (e.g. the duplication of a G in the sequence 5'-AGC-3').

A. This class of frameshift is not explained by misalignments in which a
misaligned, but Watson-Crick base paired primer can be offered as a substrate



Online LibraryNational Institute of Environmental Health ScienceAnnual report : National Institute of Environmental Health Sciences (Volume 1984 pt.1) → online text (page 10 of 60)