A General Introduction
First stages of evolution
The earliest forms of life appeared about 3.7 x 109 years ago. Development of life
on earth (biogenesis) was first a chemical, abiotic evolution, taking place in the
oceans in the presence of phosphates (XPO4), silicates (XSiO4), metal ions, an
atmosphere of nitrogen (N2), ammonia (NH3), carbon dioxide (CO2), methane (CH4),
sulfur hydrogen (H2S), hydrogen (H2) and energy sources of heat, radiation and
electric discharges. Formed were mixtures of amino acids, proteinoid microspheres
with first forms of a membrane, metabolism, growth by budding.
Biological evolution with nucleic acid chains capable of reproduction progressed in
self-organization of matter with the basic forces of evolutionary drive of mutation,
recombination, divergent development of structures and forms with specialization of
functions, selection. Formed were protobiontes, containing a short DNA strand,
which differentiated stepwise an improved metabolism, protein production, a
multifunctional membrane. The first prokaryotes of blue algae and bacteria
appeared. Following in the first evolutionary line were eukaryotes with differentiated
organelles within the cell and a membrane enclosed nucleus, containing a
chromosome set to control cell division by mitosis (start of phylogenesis). Their
oldest known chalky fossils found in oceanic sediments are about 1.5 x 109 years
old. The spreading one cell organisms took up mainly carbon and hydrogen
containing molecules in exchange for nitrogen and oxygen to radically change the
composition of the atmosphere, starting 2 x 109 years back, into the one we know
today. During the Upper Precambrium of about 9 x 108 years ago the evolutionary
rate of the diverse aquatic one cell organisms accelerated, forming multi cellular
eukaryotes with specialized cell functions. The branches of plants and animals
separated about 1 x 108 years back, introduced was sexual reproduction, further
accelerating the evolutionary rate, and set up was a heterotrophic food chain with
plants at the base, before the first forms of plant life appeared on land.

The first evolutionary line is continuously traceable in the development of genetic
materials, their proteins (phylogenetic topology) and ensuing forms of life. The first
nucleic acid chains grew by processes of base pair changes, addition, deletion,
inversion, duplication, rearrangements, activation, deactivation, in later stages by
production of catalyzing enzymes, by interaction of these factors, mostly enlarging
the overall DNA material. The development of function specific genes and proteins is
graphically demonstrated by a polygenetic tree (molecular phylogram), by branch
order and lengths, indicating their degree and distance of relation, the evolutionary
steps and the corresponding evolutionary rates. The molecular tree of lineage
delineates copy true the evolutionary tree of comparative anatomy of all plant and
animal species, stating a common ancestry of all living organisms in the bio-
chemical building blocks, the genetic code, the bio-synthesis of proteins, the cata-
lysis by enzymes, an energy metabolism with glycolysis.

The phylogenetic theory of descent serves as basis for description, denotation and
categorization (taxonomy) of all organisms. As a result of evolutionary processes,
there exist in discontinuous variability today about 5oo ooo plant and 2ooo ooo animal
species. The degree of relationship between groups, traced in a hierarchical, mono-
phyletic tree of lineage, is measured by singular, homologous, derived traits in descent
of corresponding original traits (taxon, pl. taxa). Taxonomic categories are rooted in
four kingdoms (regnum) of one cell organisms, prokaryotes, eukaryotes and mushrooms.
1

The cell nucleus
The human genetic material, the genome, is stored on 2 sets (diploid) of 23 homo-
logous chromosomes (22 autosome and 1 sex chromosome) and like all eukaryotes
confined in a cell nucleus. The hereditary information is passed on as coded se-
quences and lengths, triplets or codons, of Desoxyribonucleinacids, DNA, selected
from two purine bases guanin (G), adenin (A) and two pyrimidine bases cytosin (C),
thymin (T). The codons, yielding 43 = 64 possibilities, encode for regulative signals
and 20 essential proteins, the base group, accounting for about 2oo ooo proteins of
the human body. The codons are arranged commaless, non-overlapping. They con-
stitute a universal transcription code for all living organisms. About 6 x 107 bases,
one the primary structure, are joined by addition polymerization to form out a single
macromolecular, chromosomal strand. Two complementary strands are arranged to
a right handed, antiparallel double helix, the secondary structure. The interwinding
strands are held together by hydrogen bonds between opposite C-G and A-T base
pairs (bp). Each chromosome carries about 5o ooo genes, one the functional unit,
forming out a trait that is being passed on by Mendelian inheritance, consisting of
single copy sequences with about 1 ooo bp or polygenetically of repetitive,
multicopy sequences with 2 – 10 gene copies with 20 – 500 bp each or of
interspersed, with non-functional groups (intron) alternating, multicopy sequences.
The double helix with a diameter of 2 x 10-9 m coils itself on nucleosomes, the
tertiary structure, compressing the entire genome (chromatin) into a cell nucleus of
6 x 10-6 m in size.

Proteins
Proteins, polymeric amino acids, containing a peptide bond in the repeat unit –(
RCH (NH2) COOH )- with a molecular mass of 1o – 1oo ooo and a chain length of 5o
– 1ooo units, constitute the basic building blocks of all organisms. They represent
up to 50% of structural cell material and serve as regulative, storage, immune
active proteins. They are synthesized mainly in two steps, a process called gene
expression, by transcription within the nucleus and after transport by translation in
the cytoplasm of the cell, both proceeding over the phases of initiation, elongation
and termination. In transcription, realizing the encoded genetic information, a gene
sequence of a locally unwound chromosome string, the template, is copied base by
base onto a single stranded messenger RNA (mRNA), the matrix. In translation,
assisted by ribosomal binding sites, the matrix directs bio-synthesis, the produced
amino acid units being polymerized by addition, unidirectionally to a polypeptide
chain, followed by folding, function specific modifications, transport.

Growth processes
An organism's life cycle over the stages of zygote, embryo, youth, adult, death
(biology of development) in regular succession of generations is fueled by species
specific, somatic (non-germ) cell growth, quantitative increase of cell tissue and by
differentiation, qualitative expression of specific cell functions and organs. The
morphological changes (morphogenesis), development and arrangement of cell
populations in precise positioning and organized manner, are regulated by temporal
genes for cell specific initiators, transcriptional and translational control and by
external factors like intercellular signals, often hormones, in equilibrium with
anabolic and catabolic metabolism. Growth of a cell type is achieved by cell division
and subsequent increase of cytoplasmic volume. The periodic cell cycle proceeds in
the steps of cell division (mitosis (M)), gap (G1), synthesis (S), gap (G2). In nucleus
(karyokinesis) and cell division (cytokinesis) the chromosome set is separated to
distribute the two homologous halves to the daughter nuclei. In synthesis, the
haploid sets in each nucleus are replicated over 1o ooo replication units per chromo-
some simultaneously for a copy true, continuous passing down of the genetic
information to the next cell generation.
2

Sexual reproduction
Animal cell hybridization takes place in the sexual replication process in purpose of
reproduction, the production of new living organisms to guarantee the continuity of
the species. It proceeds in the sexual cycle in three successive stages: The male
(spermatozoa) and female (ova) germ cells grow in germ cell production (game-
togenesis) mainly out of meiosis, two cell divisions of meiosis I, proceeding in 9
phases and meiosis II, a mitotic division, proceeding in 5 phases, achieving random
assortment of chromosomes in the germ cells and a reduction of the diploid
chromosome set to one half. In cell fusion (karyogamy, conjunction of nuclei in
copulation) the gametocytes with their haploid chromosome sets are brought
together to form a fertilized egg cell (zygote), a randomly recombined diploid
chromosome complement, preserving a constant number of chromosomes. In the
third, diploid phase, the zygote develops into the embryo, the daughter generation
(ontogenesis), by successive cell divisions and development to an adult with for-
mation of sex organs to complete the sex cycle.

Hereditary traits
The genotype of an organism, the complete set of genes, determines the hereditary
traits and forms out in steps of species specific development the phenotype of the
organism (phenogenesis), the visible and empirically verifiable manifestation of a
morphological form. The phenotype is codetermined by a multitude of competing
factors like the organism's environment, by humans also by anthropological
conditions, especially social and personal environments, which change repeatedly
over a life span. A genetically hereditary trait is based on an orga-nism's identical
replication and distribution of alleles to daughter cells, on a selected bio-synthetic
pathway (gene expressivity or penetrance), on timing of gene expression of the
required gene at the required time of development from the complete genome
present in each cell (totipotency). Growth and differentiation of functions over the
stages of embryo, youth, adult (ontogenesis) form out the full complexity, capacity,
coordination and flexibility of the phenotype's hereditary traits. In humans, least
determined by its genotype are behavioral traits, because of the enormous variety
of developmental pathways of the central nervous system.

Laws of inheritance
Mendel's laws of inheritance (1865) describe the genetic recombinations of allele
pairs in sexual reproduction over successive generations, visible as hereditary traits,
where the parent generation P differs in one allele on their diploid chromosome set
with a pure homozygote wildtype a-a- and a pure homozygote mutagenic type a+a+.
The variability of the genome is passed on in new combinations, where the
progeny's ratio of genotypes is statistically predictable. Mendel's laws therefore
serve as the genetic basis for breeding technologies.
1st law of uniformity: Crossing of two pure bred homozygote strains P with the allele
combinations a-a- and a+a+ results in a first daughter generation F1, which is uniform
heterozygote in genotype 2a-a+. The trait expressed allele is called dominant, the
unexpressed recessive, codominant alleles will form out an intermediate quality or
intensity.
2nd law of segregation: Crossing of the heterozygous F1 generation results in a
second daughter generation F2 with randomly distributed allele pairs, in average a
relation of genotypes of 1:2:1 or a-a- : 2a-a+ : a+a+. The phenotypes split
correspondingly 3:1 with a trait dominant allele, 1:2:1 with trait codominant alleles.
3rd law of independent assortment: Crossing of polyhybrid Fn strains with the non-
linked allele combinations ab and cd results in a daughter generation Fn+1 with a
free combination of allele pairs, where the gene loci separate and new genotypes
and phenotypes may arise that are not present in the Fn generation.
3

Breeding technologies
Breeding techniques have been employed since prehistoric times of about 1o ooo
years. Improving plant and animal traits of quality and form like nutritional content,
yield, adaptability, resistivity, freshness, has given a major contribution to human
civilization. Selection, crossing and cultivation, utilizing genetic variability and
hereditary traits, reduce the genetic reserves, which are also depleted by
destruction of biotopes of wildtypes. Breeding (mating) systems today describe all
essential factors aside from mutation, which control population structure and
evolutionary divergence.

Breeding of a phenotype is determined by the breeding value of the trait; on the
morphological level by the kind of sex organs present, mostly dioecious, where a
partner is required to contribute the second nucleus; on the genetic level by
fertilization factors to inactivate cell specific restrictions for gamets to fuse; by
contribution of the number of chromosomes and nuclei to karyogamy; by allele
frequencies; by gene expressivity.

Main plant and animal breeding techniques comprise selection, cross, heterosis and
bio-engineered breeding:
In selection breeding a phenotype is mass selected according to its desired trait
from a mixture of a larger population for further cultivation. Directed (positive)
selection improves the degree of efficiency of a trait by elimination of one extreme,
shifting the average of the character within the population. Stabilizing (negative)
selection eliminates deviant individuals from the population, narrowing the range of
genetic variability. Disruptive selection of specific extremes leads to greater vari-
ability and to polymorphism. Through line breeding by selection over successive
generations a group of identical pure bred individuals is obtained and the chosen
trait then multiplied.
In mono- and polyhybrid crossbreeding of genetically different organisms a fusion of
alleles, surpassing incompatibility barriers, achieves in the daughter generation the
combined, desired traits in one heterozygous genotype. Genetic hybrids are mixo-
ploid combinations (mosaics) from different genera, leading to new species (chi-
mera). Through convergence breeding by recurrent selection and intercrossing the
new trait is stabilized in uniformity and consistency.
In heterosis breeding also a crossing of strains takes place, not to obtain a constant
genotype, but for the heterosis effect, where the heterozygous mix in the genome is
superior in a desired trait, which may be lacking in the P generation (hybrid vigor).
The hybrid seed can only be gained from its parent populations, as the hetero-
zygous state looses its specific mix relation by further intercrossing.

Newer breeding techniques employ in combination of mutagenic, recombinant and
hybridization DNA technologies in vitro manipulation of cell cultures in an artificial
nutrient, a semisolid or suspension medium under controlled environmental con-
ditions. They allow for example large scale breeding of life stable colonies (colony
breeding); somatic hybridization between alien gamets, bypassing fertilization
barriers, where whole, isolated, by dissolution of their walls stripped cells (proto-
plasts) of different species are fused with ones containing a nucleus or with their
nuclei removed to form a hybrid or a cybrid (cytoplasmatic hybrid); embryo split-
ting, breaking up of embryos in the 2 - 4 cell phase, cultivation and reimplantation
into two surrogate mothers; cloning, asexual reproduction of an identical, recom-
binant DNA molecule by mitosis out of a single somatic or germ cell.
4

Gen technology
Genetic engineering as discipline of molecular genetics is a part of bio-technology. It
comprises the theoretical and applied aspects of isolation, analysis, manipulation
and recombination of structural and regulative genes and their introduction, expres-
sion and multiplication in other organisms apart from naturally occurring processes.

Molecular bio-technology furnishes a significant contribution to basic research in
genetics. It developed methods for analysis of nucleotides and –sequences, their
structures, functions, reactions and products with bio-synthetic pathways and
interactions, as well as technologies for their a) isolation and identification, b) gene
mapping, c) manipulation, d) synthesis, e) ligation, f) transfer, g) transformation
and multiplication, f) test and production devices. Transformation following produc-
tion of a passenger DNA sequence, a vector system and ligation is the last step of
cloning procedures, the asexual, identical reproduction of a DNA sequence. It
opened the way for gene libraries (colony banks) and commercial production.
Applications of gene technology, the 'soft' technology, concentrate on the fields of
medicine, pharmacology, food production, human genetic diagnosis and therapy.
They expand into reproduction technologies, forensic genetics, pest control. Patents
are granted on their products and methods.
a) Methods for in vitro isolation of DNA segments are cleavage by pattern re-
cognizing restriction enzymes together with separation of DNA fragments e.g. by
blotting or polyacrylamide gel electrophoresis, separations by molecular weight with
nucleic acid and protein identification.
b) Gene mapping of DNA segments on a chromosome proceeds in orientation of
known genes by direct, fragmental DNA sequence determination or indirectly, e.g.
by radioactive marking, cleavage and identification with a gene specific DNA probe.
c) Manipulation of a DNA segment to cause a specific change in a nucleotide or –
sequence is based on the processes of DNA mutation by means of physical, che-
mical or bio-chemical incision, of DNA recombination by bio-chemical introduction,
elimination or distortion, of DNA hybridization by cell and nucleic fusion of gene-
tically close and distant (transgenic) materials.
d) Synthesis for construction of a specific DNA segment is achieved by bio-chemical
de-novo synthesis of short oligotid sequences, followed by joining of the oligo- with
polyotides, catalyzed by ligase enzymes, or by single strand synthesis through poly-
merization of complementary base pairs from a DNA matrix towards a complete
DNA duplex, added by polymerase enzymes.
e) Ligation, joining of a passenger DNA segment into the open gap of a carrier DNA
segment (replicon, vector), facilitating transformation and often with regulative se-
quences, stable gene expression, is achieved by covalent bonding, added by ligase
enzymes, which represents via indirect integration the definite step towards recom-
binant and transgenic DNA.
f) Physical transfer of the vector system as an independent unit of replication into
living host cells and cell nuclei is effected e.g. by concentration increase in form of a
precipitate or charged complex or by in vitro laser poring, a micro-injection, physi-
cally opening the cell wall.
g) Transformation (a transposition) of a recombined vector system into the host
genome aims at covalent bonding into the chromosome strands by cleavage and
joining of both ends, assisted by restriction and ligase enzymes. The passenger DNA
from in vitro cultivation is being multiplied in the nuclei of cell lines by repetitive
transformations and cell divisions, also over the stages of ontogenesis.
h) Devices for testing and automated production demand accurate, sensitive, re-
liable, fast, miniaturized measurement and process control. Bio-chemical process
parameters are taken up by bio-sensors, which contain two elements, an aggregate,
recognizing the biological information with a molecular, cell like or microorganism
bio-mass and a transducer for output of an electronic signal.
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